Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants

ABSTRACT

The invention provides isolated maize EIN3, EBF1 and EIN5 nucleic acids and encoded proteins which are associated with ethylene signaling in plants. The present invention provides methods and compositions relating to altering ethylene sensitivity in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants and antibody compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Utility patent applicationSer. No. 13/354,882 filed Jan. 20, 2012 which is a continuation of U.S.Utility patent application Ser. No. 12/274,527 filed Nov. 20, 2008, nowU.S. Pat. No. 8,129,586 issued Mar. 6, 2012 which claims priority toProvisional Application Ser. No. 60/989,368 filed Nov. 20, 2007, all ofwhich are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Plant hormones have been intensively studied for decades for theirdiverse and complex effects on the plant life. Of the five mainhormones-auxins, ethylene, abscisic acid, cytokinins andgibberellins-the molecular signaling and mode of action of ethylene hasbeen the most fully resolved. This progress was made chiefly in the1990s by the cloning of genes corresponding to mutations in ethyleneproduction and signaling.

Ethylene (C2H4) is a gaseous plant hormone that affects myriaddevelopmental processes and fitness responses in plants, such asgermination, flower and leaf senescence, fruit ripening, leafabscission, root nodulation, programmed cell death and responsiveness tostress and pathogen attack. Over the past decade, genetic screens haveidentified more than a dozen genes involved in the ethylene response inplants. Ethylene governs diverse processes in plants, and these effectsare sometimes affected by the action of other plant hormones, otherphysiological signals, and the environment, both biotic and abiotic. Forexample, it is known that cytokinin can cause ethylene like effectsthrough the action of ethylene. In addition, abscisic acid can inhibitethylene production and signaling. Auxin and ethylene are also known tocooperate in various physiological phenomena. From what is currentlyknown, in general ethylene does not appear to be strictly required forthe plant's life cycle, but it does significantly modify development andcondition response to stresses.

What is needed in the art is a means to improve agronomic performance inplants, particularly cereal crops such as maize, by modulating ethylenemediated responses in plants.

SUMMARY OF THE INVENTION

This invention involves the identification of maize genes involved inthe ethylene signal transduction pathway and the modulation of the samefor improving stress tolerance in plants. The invention relates tocharacterization and modulation of four different maize genes involvedin the ethylene pathway including, EIN3, ERF3, EBF1, EBF2 and EIN5 tocreate plants with an altered response to stress and other ethyleneinducing conditions.

Polynucleotides, related polypeptides and all conservatively modifiedvariants of the present maize sequences involved in the ethylenetransduction pathway are presented herein. Included are novel andpartial maize sequences for the ethylene signaling associated genesincluding EIN3, ERF3, EBF1, EBF2 and EIN5.

The invention also includes methods to alter the genetic composition ofcrop plants, especially maize, so that such crops can be more tolerantto stress conditions and other ethylene mediated responses. The utilityof this class of invention is then both yield enhancement and stresstolerance.

Ethylene-mediated responses include those involving: crowding tolerance,seed set and development, growth in compacted soils, flooding tolerance,maturation and senescence and disease resistance. This inventionprovides methods and compositions to effect various alterations in theethylene-mediated response in a plant that would result in improvedagronomic performance, particularly under stress.

Therefore, in one aspect, the present invention relates to an isolatednucleic acid comprising an isolated polynucleotide sequence associatedwith ethylene signaling in maize. One embodiment of the invention is anisolated polynucleotide comprising a nucleotide sequence selected fromthe group consisting of: (a) the nucleotide sequence comprising SEQ IDNO: 1 (EIN3), 3 (EBF1), 5 (EBF2), 7 (EIN5) or 9 (ERF3); (b) thenucleotide sequence encoding an amino acid sequence comprising SEQ IDNO: 2, 4, 6, 8 or 10; (c) a polynucleotide having a specified sequenceidentity to a polynucleotide encoding a polypeptide of the presentinvention; (d) a polynucleotide which is complementary to thepolynucleotide of (a); and, (e) a polynucleotide comprising a specifiednumber of contiguous nucleotides from a polynucleotide of (a) or (b).The isolated nucleic acid can be DNA.

Compositions of the invention include an isolated polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence comprising SEQ ID NO: 2, 4, 6, 8 or 10 and (b) theamino acid sequence comprising a specified sequence identity to SEQ IDNO: 2, 4, 6, 8 or 10, wherein said polypeptide has ethylene signalingactivity.

In another aspect, the present invention relates to a recombinantexpression cassette comprising a nucleic acid as described.Additionally, the present invention relates to a vector containing therecombinant expression cassette. Further, the vector containing therecombinant expression cassette can facilitate the transcription andtranslation of the nucleic acid in a host cell. The present inventionalso relates to the host cells able to express the polynucleotide of thepresent invention. A number of host cells could be used, such as but notlimited to, microbial, mammalian, plant or insect.

In yet another embodiment, the present invention is directed to atransgenic plant or plant cells, containing the nucleic acids of thepresent invention. Preferred plants containing the polynucleotides ofthe present invention include but are not limited to maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, tomatoand millet. In another embodiment, the transgenic plant is a maize plantor plant cells. Another embodiment is a transgenic seed from thetransgenic plant.

The plants of the invention can have altered responses to ethylene ascompared to a control plant. In some plants, the altered ethyleneresponse is located to a vegetative tissue, a reproductive tissue, or avegetative tissue and a reproductive tissue. Plants of the invention canhave at least one of the following phenotypes including but not limitedto: differences in crowding tolerance, seed set and development, growthin compacted soils, flooding tolerance, maturation and senescence anddisease resistance compared to non transformed plants.

Another embodiment of the invention would be plants that have beengenetically modified at a genomic locus, wherein the genomic locusencodes an ethylene signaling polypeptide of the invention.

Methods for increasing the activity of ethylene signaling polypeptidesin a plant are provided. The method can comprise introducing into theplant an ethylene signaling polynucleotide of the invention.

Methods for reducing or eliminating the level of ethylene signalingpolypeptide in the plant are also provided. The level or activity of thepolypeptide could also be reduced or eliminated in specific tissues,causing alteration in plant growth rate. Reducing the level and/oractivity of the ethylene signaling gene will lead to plants with changedresponses to the ethylene hormone.

In a further aspect, the present invention relates to a polynucleotideamplified from a Zea mays nucleic acid library using primers whichselectively hybridize, under stringent hybridization conditions, to lociwithin polynucleotides of the present invention.

DEFINITIONS

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5th edition, 1993). The terms defined below are morefully defined by reference to the specification as a whole.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology Principles and Applications, Persing, et al., Ed., AmericanSociety for Microbiology, Washington, D.C. (1993). The product ofamplification is termed an amplicon.

The term “antibody” includes reference to antigen binding forms ofantibodies. The term “antibody” frequently refers to a polypeptidesubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof which specifically bind and recognize an analyte(antigen). However, while various antibody fragments can be defined interms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments such assingle chain FV, chimeric antibodies (i.e., comprising constant andvariable regions from different species), humanized antibodies (i.e.,comprising a complementarity determining region (CDR) from a non-humansource) and heteroconjugate antibodies (e.g., bispecific antibodies).

The term “antigen” includes reference to a substance to which anantibody can be generated and/or to which the antibody is specificallyimmunoreactive. The specific immunoreactive sites within the antigen areknown as epitopes or antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition-such as amino acidsin a protein- or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i.e., substances capable of eliciting an immune response) are antigens,however some antigens, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. An antibodyimmunologically reactive with a particular antigen can be generated invivo or by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors. See, e.g., Huse, etal., (1989) Science 246:1275-1281; and Ward, et al., (1989) Nature341:544-546 and Vaughan, et al., (1996) Nature Biotech. 14:309-314.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid.

One of ordinary skill will recognize that each codon in a nucleic acid(except AUG, which is ordinarily the only codon for methionine; and UGG,which is ordinarily the only codon for tryptophan) can be modified toyield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7 or 10 alterations can be made.

Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80% or 90% of the native protein for its native substrate. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V) and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). See also, Creighton (1984) Proteins W.H. Freeman andCompany.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise interveningsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as are present in some plant, animal and fungalmitochondria, the bacterium Mycoplasma capricolumn or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed.

For example, although nucleic acid sequences of the present inventionmay be expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledons or dicotyledonsas these preferences have been shown to differ (Murray, et al., (1989)Nucl. Acids Res. 17:477-498). Thus, the maize preferred codon for aparticular amino acid may be derived from known gene sequences frommaize. Maize codon usage for 28 genes from maize plants is listed inTable 4 of Murray, et al., supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (nonsynthetic), endogenous, biologically activeform of the specified protein. Methods to determine whether a sequenceis full-length are well known in the art including such exemplarytechniques as northern or western blots, primer extension, S 1protection, and ribonuclease protection. See, e.g., Plant MolecularBiology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin(1997). Comparison to known full-length homologous (orthologous and/orparalogous) sequences can also be used to identify full-length sequencesof the present invention. Additionally, consensus sequences typicallypresent at the 5′ and 3′ untranslated regions of mRNA aid in theidentification of a polynucleotide as full-length. For example, theconsensus sequence ANNNNAUGG, where the underlined codon represents theN-terminal methionine, aids in determining whether the polynucleotidehas a complete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

By “immunologically reactive conditions” or “immunoreactive conditions”is meant conditions which allow an antibody, reactive to a particularepitope, to bind to that epitope to a detectably greater degree (e.g.,at least 2-fold over background) than the antibody binds tosubstantially any other epitopes in a reaction mixture comprising theparticular epitope. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See, Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions.

Thus, under designated immunoassay conditions, the specified antibodiesbind to an analyte having the recognized epitope to a substantiallygreater degree (e.g., at least 2-fold over background) than tosubstantially all analytes lacking the epitope which are present in thesample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, antibodies raised to the polypeptidesof the present invention can be selected from to obtain antibodiesspecifically reactive with polypeptides of the present invention. Theproteins used as immunogens can be in native conformation or denaturedso as to provide a linear epitope.

A variety of immunoassay formats may be used to select antibodiesspecifically reactive with a particular protein (or other analyte). Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine selective reactivity.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon ortransiently expressed (e.g., transfected mRNA). The term “isolated”refers to material, such as a nucleic acid or a protein, which is: (1)substantially or essentially free from components that normallyaccompany or interact with it as found in its naturally occurringenvironment. The isolated material optionally comprises material notfound with the material in its natural environment or (2) if thematerial is in its natural environment, the material has beensynthetically (non-naturally) altered by deliberate human interventionto a composition and/or placed at a location in the cell (e.g., genomeor subcellular organelle) not native to a material found in thatenvironment. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural state. Forexample, a naturally occurring nucleic acid becomes an isolated nucleicacid if it is altered, or if it is transcribed from DNA which has beenaltered, by means of human intervention performed within the cell fromwhich it originates. See, e.g., Compounds and Methods for Site DirectedMutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In VivoHomologous Sequence Targeting in Eukaryotic Cells; Zarling, et al.,PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., apromoter) becomes isolated if it is introduced by normaturally occurringmeans to a locus of the genome not native to that nucleic acid. Nucleicacids which are “isolated” as defined herein, are also referred to as“heterologous” nucleic acids.

Unless otherwise stated, the term “EIN3, ERF3, EIN5, EBF1 or EBF2nucleic acid” is a nucleic acid of the present invention and means anucleic acid comprising a polynucleotide of the present invention (a“EIN3, ERF3, EIN5, EBF1 or EBF2 polynucleotide”) encoding a EIN3, ERF3,EIN5, EBF1 or EBF2 polypeptide. A “EIN3, ERF3, EIN5, EBF1 or EBF2 gene”is a gene of the present invention and refers to a heterologous genomicform of a full-length EIN3, ERF3, EIN5, EBF1 or EBF2 polynucleotide.

As used herein, “localized within the chromosomal region defined by andincluding” with respect to particular markers includes reference to acontiguous length of a chromosome delimited by and including the statedmarkers.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes of that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, etal., Molecular Cloning-A Laboratory Manual, 2^(nd) ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollenand microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A particularly preferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or analogs thereof that havethe essential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art.

The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitization, and they may be circular, with or without branching,generally as a result of posttranslation events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific and inducible promoters constitute theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most environmental conditions.

The term “EIN3, ERF3, EIN5, EBF1 or EBF2 polypeptide” is a polypeptideof the present invention and refers to one or more amino acid sequences,in glycosylated or non-glycosylated form. The term is also inclusive offragments, variants, homologs, alleles or precursors (e.g.,preproproteins or proproteins) thereof. A “EIN3, ERF3, EIN5, EBF1 orEBF2 protein” is a protein of the present invention and comprises aEIN3, ERF3, EIN5, EBF1 or EBF2 polypeptide.

As used herein “recombinant” includes reference to a cell or vector thathas been modified by the introduction of a heterologous nucleic acid, orthat the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found in identical formwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all as a result of deliberate human intervention. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing).

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in <RTI 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Specificity is typicallythe function of post-hybridization washes, the critical factors beingthe ionic strength and temperature of the final wash solution. ForDNA/DNA hybrids, the Tm can be approximated from the equation ofMeinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: T_(m)=81.5°C.+16.6 (log M)+0.41(% GC)−0.61 (% form)−500/L; where M is the molarityof monovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % form is the percentage of formamide in thehybridization solution, and L is the length of the hybrid in base pairs.The Tm is the temperature (under defined ionic strength and pH) at which50% of a complementary target sequence hybridizes to a perfectly matchedprobe. Tm is reduced by about 1° C. for each 1% of mismatching; thus,Tm, hybridization and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the Tm can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than thethermal melting point (Tm); moderately stringent conditions can utilizea hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than thethermal melting point (Tm); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (Tm). Using the equation, hybridization and washcompositions, and desired Tm, those of ordinary skill will understandthat variations in the stringency of hybridization and/or wash solutionsare inherently described. If the desired degree of mismatching resultsin a Tm of less than 45° C. (aqueous solution) or 32° C. (formamidesolution) it is preferred to increase the SSC concentration so that ahigher temperature can be used. An extensive guide to the hybridizationof nucleic acids is found in Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); andCurrent Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

A “subject plant” or “subject plant cell” is one in which geneticalteration, such as transformation, has been affected as to a gene ofinterest or is a plant or plant cell which is descended from a plant orcell so altered and which comprises the alteration. A “control” or“control plant” or “control plant cell” provides a reference point formeasuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

In the present case, for example, changes in the ethylene response,including changes in amounts or timing of ethylene production, ethyleneactivity, ethylene distribution, ethylene signaling or ethylenerecognition or changes in plant or plant cell phenotype, such asflowering time, seed set, branching, senescence, stress tolerance orroot mass, could be measured by comparing a subject plant or plant cellto a control plant or plant cell.

As used herein, “transgenic plant” includes reference to a plant whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity” and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100 or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, (1981) Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity methodof Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCGWisconsin Package®, Version 10 (available from Accelrys Inc., 9685Scranton Road, San Diego, Calif., USA). The CLUSTAL program is welldescribed by Higgins and Sharp, (1988) Gene 73:237-244; Higgins andSharp, (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic AcidsResearch 16:10881-90; Huang, et al., (1992) Computer Applications in theBiosciences 8:155-65, and Pearson, et al., (1994) Methods in MolecularBiology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul, et al.,(1990) J. Mol. Biol. 215:403-410 and, Altschul, et al., (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information, NationalLibrary of Medicine, Building 38A, Bethesda, Md., USA. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score.

Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a word length (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a word length (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc.Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P (N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins can be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput Chem. 17:149-163) and XNU (Clayerie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values. GAP (Global Alignment Program) can also be used tocompare a polynucleotide or polypeptide of the present invention with areference sequence. GAP uses the algorithm of Needleman and Wunsch (J.Mol. Biol. 48:443-453 (1970)) to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. GAP considers all possible alignments and gap positions andcreates the alignment with the largest number of matched bases and thefewest gaps. It allows for the provision of a gap creation penalty and agap extension penalty in units of matched bases. GAP must make a profitof gap creation penalty number of matches for each gap it inserts. If agap extension penalty greater than zero is chosen, GAP must, inaddition, make a profit for each gap inserted of the length of the gaptimes the gap extension penalty. Default gap creation penalty values andgap extension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp (1989) CABIOS 5:151-153) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci.4:11-17, e. <RTI g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing Tissue distribution of Zm ERF3 expression inMPSS libraries.

FIG. 2 is a graph showing the cold-induced time-course of Zm ERF3expression in microarrays.

FIG. 3 is a diagram showing the number of genes upregulated ordown-regulated more than 5-fold both in E3 and E18 of transgenic maizeexpressing UBI::ZmERF3.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention provides, among other things, compositions andmethods for modulating (i.e., increasing or decreasing) the level ofpolynucleotides and polypeptides of the present invention in plants. Inparticular, the polynucleotides and polypeptides of the presentinvention can be expressed temporally or spatially, e.g., atdevelopmental stages, in tissues, and/or in quantities, which areuncharacteristic of non-recombinantly engineered plants. Thus, thepresent invention provides utility in such exemplary applications asprovided below.

EIN3 is a nuclear transcription factor that binds to ethylene responseelements present in the promoters of ethylene response factors, such asERF1 or ERF3. Thus down regulation of EIN3 will reduce ethylenesensitivity.

EBF1 and EBF2 are F-box proteins that bind to EIN3 and target the EIN3protein for degradation through the ubiquitin/proteasome pathway. Thusoverexpression of EBF1 and EBF2 will decrease ethylene sensitivity. EIN3protein levels rapidly increase in response to ethylene and thisresponse requires several ethylene-signaling pathway componentsincluding the ethylene receptors (ETR1 and EIN4), CTR1, EIN2, EIN5 andEIN6. In the absence of ethylene, EIN3 is quickly degraded through aubiquitin/proteasome pathway mediated by EBF1 and EBF2.

EIN5 has endonuclease activity on EBF1 and EBF2 transcripts and thusantagonizes the negative feedback on EIN3 by promoting EBF1 and EBF2mRNA decay. Thus down regulation of EIN5 will reduce ethylenesensitivity.

Changes in the ethylene response may include one or more changes inamount or timing of ethylene production, ethylene activity, ethylenedistribution, ethylene signaling and ethylene recognition, which mayresult in phenotypic changes such as, for example, altered floweringtime, seed set, branching, senescence, stress tolerance, or rootformation,

In certain embodiments the nucleic acid constructs of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes, and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; that is, certain combinations may be created for modulationof gene expression affecting ethylene response. Other combinations maybe designed to produce plants with a variety of desired traits, such asthose described elsewhere herein.

Crowding Tolerance

The agronomic performance of crop plants is often a function of how wellthey tolerate planting density. The stress of overcrowding can be due tosimple limitations of nutrients, water, and sunlight. Crowding stressmay also be due to enhanced contact between plants. Plants often respondto physical contact by slowing growth and thickening their tissues.

Ethylene has been implicated in plant crowding tolerance. For example,ethylene insensitive tobacco plants did not slow growth when contactingneighboring plants (Knoester, et al., (1998) PNAS USA 95:1933-1937).There is also evidence that ethylene, and the plant's response to it, isinvolved in water deficit stress, and that ethylene may be causingchanges in the plant that limit its growth and aggravate the symptoms ofdrought stress beyond the loss of water itself.

The present invention provides for decreasing ethylene sensitivity in aplant, in particular cereals such as maize, by modulating the expressionor activity of one or more of EIN3, ERF3, EBF1, EBF2 or EIN5 genes orgene products to promote tolerance of close spacing with reduced stressand minimized yield loss.

Seed Set and Development in Maize

Ethylene plays a number of roles in seed development. For example, inmaize ethylene is linked to programmed cell death of developingendosperm cells (Young, et al., (1997) Plant Physio. 115:737-751). Inaddition, ethylene is linked to kernel abortion, such as occurs at thetips of ears, especially in plants grown under stressful conditions(Cheng and Lur, (1996) Physiol. Plant 98:245-252). Reduced kernel seedset is of course a contributor to reduced yields. Consequently, thepresent invention provides plants, in particular maize plants that havereduced ethylene action by altering expression of genes involved in theethylene response.

Growth in Compacted Soils

Plant growth is affected by the density and compaction of soils. Denser,more compacted soils typically result in poorer plant growth. The trendin agriculture towards more minimal till planting and cultivationpractices, with the goal of soil and energy conservation, is increasingthe need for crop plants that can perform well under these conditions.

Ethylene is well-known to affect plant growth and development, and oneeffect of ethylene is to promote tissue thickening and growthretardation when encountering mechanical stress, such as compactedsoils. This can affect both the roots and shoots. This effect ispresumably adaptive in some circumstances in that it results instronger, more compact tissues that can force their way through oraround, obstacles such as compacted soils. However, in such conditions,the production of ethylene and the activation of the ethylene pathwaymay exceed what is needed for adaptive accommodation to the mechanicalstress of the compacted soils. Any resulting unnecessary growthinhibition would be an undesired agronomic result.

The present invention provides for decreasing ethylene sensitivity in aplant, in particular cereals such as maize, by modulating the expressionor activity of one or more of EIN3, ERF3, EBF1, EBF2 or EIN5 genes orgene products. Such modulated plants grow and germinate better incompacted soils, resulting in higher stand counts, the herald of higheryields.

Flooding Tolerance

Flooding and water-logged soils causes substantial losses in crop yieldeach year around the world. Flooding can be both widespread or local,transitory or prolonged. Ethylene has been implicated in floodingmediated damage. In fact, in flooded conditions ethylene production canrise. There are two main reasons for this rise: 1) under such floodedconditions, which creates hypoxia, plants produce more ethylene, and 2)under flooded conditions the diffusion of ethylene away from the plantis slowed, because ethylene is minimally soluble in water, resulting ina rise of intra-plant ethylene levels.

In rice, submergence tolerance is known to be imparted through ethylenesignaling (Perata and Voesenek, (2007) Trends Plant Sci 12(2):43-46).

Ethylene in flooded maize roots can also inhibit gravitropism, which isnormally adaptive during germination in that it orients the roots downand the shoots up. Gravitropism is a factor in determining rootarchitecture, which in turn plays an important role in soil resourceacquisition. Manipulation of ethylene levels could be used to impactroot angle for drought tolerance, flood tolerance, greater standabilityand/or improved nutrient uptake. For example, a root growing at a moreerect angle (steeper) would likely grow more deeply in soil and thusobtain water at greater depths, improving drought tolerance. In theabsence of drought stress a converse argument could be made for moreefficient root uptake of nutrients and water in the upper layers of thesoil profile, by roots which are more parallel to the soil surface. Ingeneral, roots that have a angle nearer that of vertical (steep) arealso more susceptible to root lodging than roots with a shallow angle(parallel to the surface) that can be more root lodging resistant.

In addition to inhibition of gravitropism, it is likely that ethyleneevolution in flooded conditions inhibits growth, especially of roots.Such inhibition will likely contribute to poor plant growth overall, andconsequently is a disadvantageous agronomic trait.

The present invention provides for decreasing ethylene sensitivity in aplant, in particular cereals such as maize, by modulating the expressionor activity of one or more of EIN3, ERF3, EBF1, EBF2 or EIN5 genes orgene products. Such plants should grow and germinate better in floodedconditions or water-logged soils, resulting in higher stand counts.

Plant Maturation and Senescence

Ethylene is known to be involved in controlling senescence, fruitripening, and abscission. The role of ethylene in fruit ripening iswell-established and industrially applied. It is expected that ethyleneunderproduction/insensitivity would result in slower seed maturation orfruit ripening, and the converse would result in more rapid seedmaturation or fruit ripening. Abscission is primarily studied for dicotplants and apparently has little application to monocots such ascereals. Ethylene mediated senescence also is mostly studied in dicots,but control of senescence is agronomically important for both dicot andmonocot crop species. Ethylene insensitivity can delay, but not arrest,senescence. The senescence process mediated by ethylene bears somesimilarities to the cell death process in disease symptoms and inabscission zones. Controlling ethylene sensitivity, as through thecontrol of one or more of the ERF3, EIN3, EBF1, EBF2, EIN5 genes couldresult in modulation of maturity rates for crop plants such as maize.

The present invention provides for decreasing ethylene sensitivity in aplant, in particular cereals such as maize, by modulating the expressionor activity of one or more of EIN3, ERF3, EBF1, EBF2 or EIN5 genes whichmay contribute to a later maturing plant, which is desirable for placingcrop varieties in different maturity zones.

Tolerance to Other Abiotic Stresses

Many stresses on plants induce production of ethylene (see, Morgan andDrew, (1997) Physiol. Plant 100:620-630). These stresses can be, forexample, cold, heat, wounding, pollution, drought, and hypersalinity.Mechanical impedance (soil compaction) and flooding stresses wereaddressed above. It appears that several of these stresses operatethrough common mechanisms, such as water deficit. Clearly drought causeswater deficit, crowding stress may also cause water deficit.Additionally, in maize, chilling can cause an elevation in ethyleneproduction and activity, and this induction is apparently due tochilling causing water deficit in cells (Janowaik and Dorffling, (1995)J. Plant Physiol. 147:257-262).

Some of the ethylene production following stresses may serve an adaptivepurpose by regulating ethylene-mediated processes in the plant thatresult in a plant reorganized in such manner to better acclimate to thestress encountered. However, there is also evidence that ethyleneproduction during stress can result in an aggravation of negativesymptoms resulting from the stress, such as yellowing, tissue death andsenescence.

To the extent that ethylene production during stress causes or augmentsnegative stress-related symptoms, it would be desirable to create a cropplant that is less sensitive to the ethylene. Towards that end, thepresent invention provides for decreasing ethylene sensitivity in aplant, in particular cereals such as maize, by modulating the expressionor activity of one or more of EIN3, ERF3, EBF1, EBF2 or EIN5 genes orgene products to create plants that are less able to produce ethylenemediated effects.

Disease Resistance

Crop plants can be susceptible to a wide variety of pathogens, whetherviruses, bacteria, fungi or insects. This susceptibility results inlarge crop yield losses annually worldwide. Crop breeders haveendeavored to breed more resistant or tolerant varieties which canwithstand pathogen attack. Additional genetic engineering strategiesseek the same end. In many plant-pathogen interactions the symptoms ofdisease, most often tissue necrosis and resulting poor plant growth, isknown to be the result of an active plant defense response to thepathogen. That is, the symptoms are caused directly by the plant and notsimply by the pathogen. From among the list of all crop plants and theirpotential list of pathogens, resistance is the rule, and susceptibilitythe exception. Susceptible interactions are often thought to result froman improper or insufficient activation defense by the plant that resultsin increased symptom development and an inability to contain thepathogen.

Ethylene is known to be associated with plant pathogen defense systems.Many pathogenesis related genes are induced in expression at the levelof mRNA by ethylene. The trend in our understanding of the role ofethylene in plant pathogen defense is towards ethylene and ethylenemediated effects being viewed as principally part of the downstreamreactions to pathogen attack, as in symptom development. Ethylene seemsto be involved in the plant's response to the stress of pathogen attackand in tissue damage inflicted by the pathogen. In a susceptibleinteraction ethylene may actually promote tissue damage. Consequently insuch situations blocking ethylene production or action may actuallyresult in less tissue damage, that is, more apparent resistance, eventhough the pathogen is compatible with the plant. Blocking ethyleneaction is known to either result in more susceptibility (e.g., Knoester,et al., (1988)) or more resistance (e.g., Lund, et al., (1998) PlantCell 10:371-382), which indicates that the role of ethylene action iscomplex, as is to be expected, for it depends upon the interactions ofdiverse plants and pathogens.

The present invention provides for the use of one or more of EIN3, ERF3,EBF1, EBF2 or EIN5 genes to affect enhanced resistance to plantpathogens, in particular for monocots such as maize.

For most applications this will involve the reduction in ethylenesignaling by modulating the expression or activity of EIN3, ERF3, EBF1,EBF2 or EIN5 genes or gene products, with the goal of causing plantsthat responds less to ethylene and thereby plants that are less prone totissue damage following pathogen infection.

It is recognized that for some pathogens, ethylene signaling may benecessary for achieving substantial resistance. This can be handled bylinking a functional ethylene sensitivity gene to a pathogen-induciblepromoter, in particular to a promoter whose induction is preferentiallyresponsive to the pathogen or pathogens for which plant ethylenesignaling is desired for achievement of active resistance.

Plant Transformation

The generation of transgenic plants is central to crop plant geneticengineering strategies. Transgenesis typically involves the introductionof exogenous DNA into the plants cells via a variety of methods, such asparticle bombardment or agrobacterium infection, which is usuallyfollowed by tissue culture and plant regeneration. Transgenic plantproduction remains a costly and rate limiting step in geneticengineering, especially for many of the most economically important cropplants, such as the cereals, like maize.

Improving the efficiency of this process is therefore of greatimportance.

It has been accepted for a long time that ethylene action has negativeconsequences for plant transformation. As a result various approaches tobind, trap or otherwise block the accumulation of ethylene are employedin transformation and tissue culture (see, Songstad, et al., (1991)Plant Cell Reports 9:694-702). The particle bombardment method causessubstantial tissue/cell damage, and such damage is known to elicitethylene accumulation. Moreover, in most tissue culture methods, sometissue grows better than others, as is designed in chemical selection oftransformants. Such dying tissue can emit ethylene and cause inhibitionof positive transformants. Aggravating these effects is the confinementof plant tissues in containers for the purpose of tissue regeneration,that can result in the accumulation of ethylene, also causing growthretardation. As ethylene is known to reduce tissue growth rates and evenadvance cell/tissue death, having a means to block or minimize ethyleneaction during transformation is desired.

Consequently, the present invention also provides for the use of anEIN3, ERF3, EBF1, EBF2 or EIN5 gene to create transient or stablereductions in ethylene action by diminishing the expression and/oractivity of one or more of the EIN3, ERF3, EBF1, EBF2 or EIN5 genes.

Other Utilities

The present invention also provides isolated nucleic acids comprisingpolynucleotides of sufficient length and complementarity to a gene ofthe present invention to use as probes or amplification primers in thedetection, quantitation or isolation of gene transcripts. For example,isolated nucleic acids of the present invention can be used as probes indetecting deficiencies in the level of mRNA in screenings for desiredtransgenic plants, for detecting mutations in the gene (e.g.,substitutions, deletions or additions), for monitoring upregulation ofexpression or changes in enzyme activity in screening assays ofcompounds, for detection of any number of allelic variants(polymorphisms), orthologs, or paralogs of the gene, or for sitedirected mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No.5,565,350). The isolated nucleic acids of the present invention can alsobe used for recombinant expression of their encoded polypeptides, or foruse as immunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue, or plant. Attachment ofchemical agents which bind, intercalate, cleave and/or cross-link to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

The present invention also provides isolated proteins comprising apolypeptide of the present invention (e.g., preproenzyme, proenzyme orenzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function, or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species, or for purification ofpolypeptides of the present invention.

The isolated nucleic acids and polypeptides of the present invention canbe used over a broad range of plant types, particularly monocots such asthe species of the family Gramineae including Hordeum, Secale, Tritium,Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays). The isolated nucleicacid and proteins of the present invention can also be used in speciesfrom the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus,Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,Petunia, Digitalis, Majorana, Clahorium, Helianthus, Lactuca, Bromus,Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum,Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia,Glycine, Pisum, Phaseolus, Lolium, Oryza and Avena.

Nucleic Acids

The present invention provides, among other things, isolated nucleicacids of RNA, DNA, and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

A polynucleotide of the present invention is inclusive of:

-   -   (a) a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 4,        6, 8 or 10, and conservatively modified and polymorphic variants        thereof, including exemplary polynucleotides of SEQ ID NOS: 1        (EIN3), 3 (EBF1), 5 (EBF2), 7 (EIN5) or 9 (ERF3);    -   (b) an isolated polynucleotide which is the product of        amplification from a plant nucleic acid library using primer        pairs which selectively hybridize under stringent conditions to        loci within a polynucleotide of the present invention;    -   (c) an isolated polynucleotide which selectively hybridizes to a        polynucleotide of (a) or (b);    -   (d) an isolated polynucleotide having a specified sequence        identity with polynucleotides of (a), (b) or (c);    -   (e) an isolated polynucleotide encoding a protein having a        specified number of contiguous amino acids from a prototype        polypeptide, wherein the protein is specifically recognized by        antisera elicited by presentation of the protein and wherein the        protein does not detectably immunoreact to antisera which has        been fully immunosorbed with the protein;    -   (f) complementary sequences of polynucleotides of (a), (b),        (c), (d) or (e); and    -   (g) an isolated polynucleotide comprising at least a specific        number of contiguous nucleotides from a polynucleotide of (a),        (b), (c), (d), (e) or (f);    -   (h) an isolated polynucleotide from a full-length enriched cDNA        library having the physico-chemical property of selectively        hybridizing to a polynucleotide of (a), (b), (c), (d), (e), (f)        or (g);    -   (i) an isolated polynucleotide made by the process of: 1)        providing a full-length enriched nucleic acid library, 2)        selectively hybridizing the polynucleotide to a polynucleotide        of (a), (b), (c), (d), (e), (f), (g) or (h), thereby isolating        the polynucleotide from the nucleic acid library.

A. Polynucleotides Encoding a Polypeptide of the Present Invention

As indicated in (a), above, the present invention provides isolatednucleic acids comprising a polynucleotide of the present invention,wherein the polynucleotide encodes a polypeptide of the presentinvention. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Thus, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and is within the scope of the present invention.Accordingly, the present invention includes polynucleotides of thepresent invention and polynucleotides encoding a polypeptide of thepresent invention.

B. Polynucleotides Amplified from a Plant Nucleic Acid Library

As indicated in (b), above, the present invention provides an isolatednucleic acid comprising a polynucleotide of the present invention,wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library.

Nucleic acid amplification conditions for each of the variety ofamplification methods are well known to those of ordinary skill in theart. The plant nucleic acid library can be constructed from a monocotsuch as a cereal crop. Exemplary cereals include corn, sorghum, alfalfa,canola, wheat or rice. The plant nucleic acid library can also beconstructed from a dicot such as soybean. Zea mays lines B73, PHRE1,A632, BMP2#10, W23 and Mol7 are known and publicly available. Otherpublicly known and available maize lines can be obtained from the MaizeGenetics Cooperation (Urbana, Ill.).

Wheat lines are available from the Wheat Genetics Resource Center(Manhattan, Kans.). The nucleic acid library may be a cDNA library, agenomic library, or a library generally constructed from nucleartranscripts at any stage of intron processing. cDNA libraries can benormalized to increase the representation of relatively rare cDNAs. Inoptional embodiments, the cDNA library is constructed using an enrichedfull-length cDNA synthesis method. Examples of such methods includeOligo-Capping (Maruyama and Sugano, (1994) Gene 138:171-174),Biotinylated CAP Trapper (Carninci, et al., (1996) Genomics 37:327-336)and CAP Retention Procedure (Edery, et al., (1995) Molecular andCellular Biology 15:3363-3371). Rapidly growing tissues or rapidlydividing cells are preferred for use as an mRNA source for constructionof a cDNA library. Growth stages of corn are described in “How a CornPlant Develops, “Special Report No. 48, Iowa State University of Scienceand Technology Cooperative Extension Service, Ames, Iowa, ReprintedFebruary 1993.

A polynucleotide of this embodiment (or subsequences thereof) can beobtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, inPCR Protocols: A Guide to Methods and Applications, Innis, et al., Eds.(Academic Press, Inc., San Diego), pp. 28-38 (1990)); see also, U.S.Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995); Frohman and Martin, (1989) Techniques 1:165.

Optionally, the primers are complementary to a subsequence of the targetnucleic acid which they amplify but may have a sequence identity rangingfrom about 85% to 99% relative to the polynucleotide sequence which theyare designed to anneal to. As those skilled in the art will appreciate,the sites to which the primer pairs will selectively hybridize arechosen such that a single contiguous nucleic acid can be formed underthe desired nucleic acid amplification conditions. The primer length innucleotides is selected from the group of integers consisting of from atleast 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40or 50 nucleotides in length. Those of skill will recognize that alengthened primer sequence can be employed to increase specificity ofbinding (i.e., annealing) to a target sequence. A non-annealing sequenceat the 5′ end of a primer (a “tail”) can be added, for example, tointroduce a cloning site at the terminal ends of the amplicon.

The amplification products can be translated using expression systemswell known to those of skill in the art. The resulting translationproducts can be confirmed as polypeptides of the present invention by,for example, assaying for the appropriate catalytic activity (e.g.,specific activity and/or substrate specificity) or verifying thepresence of one or more epitopes which are specific to a polypeptide ofthe present invention. Methods for protein synthesis from PCR derivedtemplates are known in the art and available commercially. See, e.g.,Amersham Life Sciences, Inc, Catalog '97, p. 354.

C. Polynucleotides which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated in (c), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides selectively hybridize, under selectivehybridization conditions, to a polynucleotide of sections (A) or (B) asdiscussed above. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate or amplify partial or full-length clones in a deposited library.

In some embodiments, the polynucleotides are genomic or cDNA sequencesisolated or otherwise complementary to a cDNA from a dicot or monocotnucleic acid library.

Exemplary species of monocots and dicots include, but are not limitedto: maize, canola, soybean, cotton, wheat, sorghum, sunflower, alfalfa,oats, sugar cane, millet, barley and rice. The cDNA library comprises atleast 50% to 95% full-length sequences (for example, at least 50%, 60%,70%, 80% 90% or 95% full-length sequences). The cDNA libraries can benormalized to increase the representation of rare sequences. See, e.g.,U.S. Pat. No. 5,482,845. Low stringency hybridization conditions aretypically, but not exclusively, employed with sequences having a reducedsequence identity relative to complementary sequences. Moderate and highstringency conditions can optionally be employed for sequences ofgreater identity. Low stringency conditions allow selectivehybridization of sequences having about 70% to 80% sequence identity andcan be employed to identify orthologous or paralogous sequences.

D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides have a specified identity at the nucleotidelevel to a polynucleotide as disclosed above in sections (A), (B) or(C), above. Identity can be calculated using, for example, the BLAST,CLUSTALW or GAP algorithms under default conditions. The percentage ofidentity to a reference sequence is at least 60% and, rounded upwards tothe nearest integer, can be expressed as an integer selected from thegroup of integers consisting of from 60 to 99. Thus, for example, thepercentage of identity to a reference sequence can be at least 70%, 75%,80%, 85%, 90% or 95%.

Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B) or (C). Thus, these polynucleotidesencode a first polypeptide which elicits production of antiseracomprising antibodies which are specifically reactive to a secondpolypeptide encoded by a polynucleotide of (A), (B) or (C). However, thefirst polypeptide does not bind to antisera raised against itself whenthe antisera has been fully immunosorbed with the first polypeptide.Hence, the polynucleotides of this embodiment can be used to generateantibodies for use in, for example, the screening of expressionlibraries for nucleic acids comprising polynucleotides of (A), (B) or(C), or for purification of, or in immunoassays for, polypeptidesencoded by the polynucleotides of (A), (B) or (C). The polynucleotidesof this embodiment comprise nucleic acid sequences which can be employedfor selective hybridization to a polynucleotide encoding a polypeptideof the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure.

Antibody screening of peptide display libraries is well known in theart. The displayed peptide sequences can be from 3 to 5000 or more aminoacids in length, frequently from 5100 amino acids long, and often fromabout 8 to 15 amino acids long. In addition to direct chemical syntheticmethods for generating peptide libraries, several recombinant DNAmethods have been described. One type involves the display of a peptidesequence on the surface of a bacteriophage or cell. Each bacteriophageor cell contains the nucleotide sequence encoding the particulardisplayed peptide sequence. Such methods are described in PCT PatentPublication Numbers 1991/17271, 1991/18980, 1991/19818 and 1993/08278.Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Numbers 1992/05258, 1992/14843 and 1997/20078. See also,U.S. Pat. Nos. 5,658,754 and 5,643,768. Peptide display libraries,vectors, and screening kits are commercially available from suchsuppliers as Invitrogen (Carlsbad, Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

As indicated in (e), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides encode a protein having a subsequence ofcontiguous amino acids from a prototype polypeptide of the presentinvention such as are provided in (a), above. The length of contiguousamino acids from the prototype polypeptide is selected from the group ofintegers consisting of from at least 10 to the number of amino acidswithin the prototype sequence. Thus, for example, the polynucleotide canencode a polypeptide having a subsequence having at least 10, 15, 20,25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototypepolypeptide. Further, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera has been fully immunosorbed withthe prototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

In a preferred assay method, fully immunosorbed and pooled antiserawhich is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen.

Accordingly, the proteins of the present invention embrace allelicvariants, conservatively modified variants, and minor recombinantmodifications to a prototype polypeptide.

A polynucleotide of the present invention optionally encodes a proteinhaving a molecular weight as the non-glycosylated protein within 20% ofthe molecular weight of the full-length non-glycosylated polypeptides ofthe present invention. Molecular weight can be readily determined bySDS-PAGE under reducing conditions. Optionally, the molecular weight iswithin 15% of a full length polypeptide of the present invention, morepreferably within 10% or 5%, and most preferably within 3%, 2% or 1% ofa full length polypeptide of the present invention. Optionally, thepolynucleotides of this embodiment will encode a protein having aspecific enzymatic activity at least 50%, 60%, 80% or 90% of a cellularextract comprising the native, endogenous full-length polypeptide of thepresent invention.

Further, the proteins encoded by polynucleotides of this embodiment willoptionally have a substantially similar affinity constant (Km) and/orcatalytic activity (i.e., the microscopic rate constant, kcat) as thenative endogenous, full-length protein. Those of skill in the art willrecognize that kcat/Km value determines the specificity for competingsubstrates and is often referred to as the specificity constant.Proteins of this embodiment can have akcat/Km value at least 10% of afull-length polypeptide of the present invention as determined using theendogenous substrate of that polypeptide. Optionally, the kcat/Km valuewill be at least 20%, 30%, 40%, 50% and most preferably at least 60%,70%, 80%, 90% or 95% the kcat/Km value of the full-length polypeptide ofthe present invention.

Determination of kcat, Km, and kcat/Km can be determined by any numberof means well known to those of skill in the art. For example, theinitial rates (i.e., the first 5% or less of the reaction) can bedetermined using rapid mixing and sampling techniques (e.g.,continuous-flow, stopped-flow or rapid quenching techniques), flashphotolysis or relaxation methods (e.g., temperature jumps) inconjunction with such exemplary methods of measuring asspectrophotometry, spectrofluorimetry, nuclear magnetic resonance orradioactive procedures. Kinetic values are conveniently obtained using aLineweaver Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)

As indicated in (f), above, the present invention provides isolatednucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil.

G. Polynucleotides Which are Subsequences of the Polynucleotides of(A)-(F)

As indicated in (g), above, the present invention provides isolatednucleic acids comprising polynucleotides which comprise at least 15contiguous bases from the polynucleotides of sections (A) through (F) asdiscussed above. The length of the polynucleotide is given as an integerselected from the group consisting of from at least 15 to the length ofthe nucleic acid sequence from which the polynucleotide is a subsequenceof. Thus, for example, polynucleotides of the present invention areinclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50,60, 75 or 100 contiguous nucleotides in length from the polynucleotidesof (A)-(F). Optionally, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

Subsequences can be made by in vitro synthetic, in vitro biosynthetic orin vivo recombinant methods. In optional embodiments, subsequences canbe made by nucleic acid amplification. For example, nucleic acid primerswill be constructed to selectively hybridize to a sequence (or itscomplement) within, or co-extensive with, the coding region.

The subsequences of the present invention can comprise structurallibraries are known in the art and discussed briefly below. The cDNAlibrary comprises at least 50% to 95% full-length sequences (forexample, at least 50%, 60%, 70%, 80%, 90% or 95% full-length sequences).The cDNA library can be constructed from a variety of tissues from amonocot or dicot at a variety of developmental stages. Exemplary speciesinclude maize, wheat, rice, canola, soybean, cotton, sorghum, sunflower,alfalfa, oats, sugar cane, millet, barley and rice. Methods ofselectively hybridizing, under selective hybridization conditions, apolynucleotide from a full-length enriched library to a polynucleotideof the present invention are known to those of ordinary skill in theart. Any number of stringency conditions can be employed to allow forselective hybridization. In optional embodiments, the stringency allowsfor selective hybridization of sequences having at least 70%, 75%, 80%,85%, 90%, 95% or 98% sequence identity over the length of the hybridizedregion. Full-length enriched cDNA libraries can be normalized toincrease the representation of rare sequences.

I. Polynucleotide Products Made by a cDNA Isolation Process

As indicated in (I), above, the present invention provides an isolatedpolynucleotide made by the process of: 1) providing a full-lengthenriched nucleic acid library, 2) selectively hybridizing thepolynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G) or (H) as discussed above, and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.

Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis and thelike.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot such as corn, rice or wheat or a dicot such as soybean.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexahistidine marker sequence provides a convenient meansto purify the proteins of the present invention. A polynucleotide of thepresent invention can be attached to a vector, adapter, or linker forcloning and/or expression of a polynucleotide of the present invention.Additional sequences may be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide or to improve the introduction ofthe polynucleotide into a cell. Typically, the length of a nucleic acidof the present invention less the length of its polynucleotide of thepresent invention is less than 20 kilobase pairs, often less than 15 kb,and frequently less than 10 kb. Use of cloning vectors, expressionvectors, adapters and linkers is well known and extensively described inthe art. For a description of various nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1999 (La Jolla, Calif.) andAmersham Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).

A. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA, andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); andCurrent Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

A1. Full-Length Enriched cDNA Libraries

A number of cDNA synthesis protocols have been described which provideenriched full-length cDNA libraries. Enriched full-length cDNA librariesare constructed to comprise at least 60%, and more preferably at least70%, 80%, 90% or 95% full-length inserts amongst clones containinginserts. The length of insert in such libraries can be at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate insertsof these sizes are known in the art and available commercially. See,e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12kb cloning capacity). An exemplary method of constructing a greater than95% pure full-length cDNA library is described by Carninci, et al.,(1996) Genomics 37:327-336. Other methods for producing full-lengthlibraries are known in the art. See, e.g., Edery, et al., (1995) Mol.Cell Biol. 15(6):3363-3371 and PCT Application WO 1996/34981.

A2 Normalized or Subtracted cDNA Libraries

A non-normalized cDNA library represents the mRNA population of thetissue it was made from. Since unique clones are out-numbered by clonesderived from highly expressed genes their isolation can be laborious.Normalization of a cDNA library is the process of creating a library inwhich each clone is more equally represented. Construction of normalizedlibraries is described in Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711;Patanjali, et al., (1991) Proc. Natl. Acad. USA 88:1943-1947; U.S. Pat.Nos. 5,482,685, 5,482,845 and 5,637,685. In an exemplary methoddescribed by Soares, et al., normalization resulted in reduction of theabundance of clones from a range of four orders of magnitude to a narrowrange of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA,91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. In this procedure, cDNA prepared from onepool of mRNA is depleted of sequences present in a second pool of mRNAby hybridization. The cDNA: mRNA hybrids are removed and the remainingun-hybridized cDNA pool is enriched for sequences unique to that pool.See, Foote, et al., in, Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991)Technique 3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res.16(22):10937; Current Protocols in Molecular Biology, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995); andSwaroop, et al., (1991) Nucl. Acids Res. 19(8):1954. cDNA subtractionkits are commercially available. See, e.g., PCR-Select (Clontech, PaloAlto, Calif.).

To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g., using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

The cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have been described. Wilfinger, et al.,describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.Bio Techniques 22(3):481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-99; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage, et al.,(1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, (1981) Tetra.Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter, et al., (1984) Nucleic Acids Res.12:6159-6168; and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is best employed for sequences of about 100bases or less, longer sequences may be obtained by the ligation ofshorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polypeptide of the present invention,for example a cDNA or a genomic sequence encoding a full lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

For example, plant expression vectors may include: (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,theGRP1-8 promoter and other transcription initiation regions fromvarious plant genes known to those of skill.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adhl promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds or flowers. Exemplary promoters includethe anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051), glob-1 promoter, and gamma-zein promoter. The operation of apromoter may also vary depending on its location in the genome. Thus, aninducible promoter may become fully or partially constitutive in certainlocations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue. Thus, in someembodiments, the nucleic acid construct will comprise a promoterfunctional in a plant cell, such as in Zea mays, operably linked to apolynucleotide of the present invention. Promoters useful in theseembodiments include the endogenous promoters driving expression of apolypeptide of the present invention.

A number of promoters can be used in the practice of the invention,including the native promoter of a polynucleotide sequence of interest.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, inducible, tissue-preferred orother promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell,et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990)Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol.Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol.18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S.Pat. No. 5,659,026), dMMV (double-enhanced version of the mirabilismosaic virus promoter; see, Dey and Maiti (1999) Plant Molecular Biology40(5):771-782), LESVBV (enhanced strawberry vein banding virus promoter;see, US Patent Application Publication Number 2002/0182593) and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142 and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters includeYamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al.,(1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol.Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res.6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341;Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, etal., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994)Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco, et al., (1993) Plant Mol. Biol.23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression. See, also, US Patent Application Publication Number2003/0074698, herein incorporated by reference.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988)Nucl. Acid Res. 16:4732; Mitra, et al., (1994) Plant Molecular Biology26:35-93; Kayaya, et al., (1995) Molecular and General Genetics248:668-674; and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590. Senecence regulated promoters are also of use, suchas, SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:459-466). See,also, U.S. Pat. No. 5,689,042 herein incorporated by reference.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire, et al., (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger, et al.,(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, etal., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi, (1991) describe their analysisof the promoters of the highly expressed roIC and rolD root-inducinggenes of Agrobacterium rhizogenes (see, Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri, et al., (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see, EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); roIBpromoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691; and theCRWAQ81 root-preferred promoter with the ADH first intron (US PatentApplication Publication Number 2005/0097633). See also, U.S. Pat. Nos.5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and5,023,179.

“Seed-preferred” promoters refers to those promoters active during seeddevelopment and may include expression in seed initials or relatedmaternal tissue. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); milps (myo-inositol-1-phosphate synthase) (see, WO 00/11177 andU.S. Pat. No. 6,225,529; herein incorporated by reference). Gamma-zeinis an endosperm-specific promoter. Globulin-1 (Glob-1) is arepresentative embryo-specific promoter. For dicots, seed-specificpromoters include, but are not limited to, bean β-phaseolin, napin,β-conglycinin, soybean lectin, cruciferin and the like. For monocots,seed-specific promoters include, but are not limited to, maize 15 kDazein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1 andshrunken 2. See also, WO 2000/12733, where seed-preferred promoters fromend1 and end2 genes are disclosed; herein incorporated by reference.Additional embryo specific promoters are disclosed in Sato, et al.,(1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al., (1997) PlantJ 12:235-46; and Postma-Haarsma, et al., (1999) Plant Mol. Biol.39:257-71. Additional endosperm specific promoters are disclosed inAlbani, et al., (1984) EMBO 3:1405-15; Albani, et al., (1999) Theor.Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena,et al., (1998) The Plant Journal 116:53-62 and Wu, et al., (1998) PlantCell Physiology 39:885-889.

Also of interest are promoters active in meristem regions, such asdeveloping inflorescence tissues, and promoters which drive expressionat or about the time of anthesis or early kernel development. This mayinclude, for example, the maize Zag promoters, including Zag1 and Zag2(see, Schmidt, et al., (1993) The Plant Cell 5:729-37; GenBank X80206;Theissen, et al., (1995) Gene 156:155-166; and U.S. patent applicationSer. No. 10/817,483); maize Zap promoter (also known as ZmMADS; U.S.patent application Ser. No. 10/387,937; WO 03/078590); maize ck×1-2promoter (US Patent Application Publication Number 2002/0152500 A1; WO2002/0078438); maize eep1 promoter (U.S. patent application Ser. No.10/817,483); maize end2 promoter (U.S. Pat. No. 6,528,704 and U.S.patent application Ser. No. 10/310,191); maize lec1 promoter (U.S.patent application Ser. No. 09/718,754); maize F3.7 promoter(Baszczynski, et al., (1997) Maydica 42:189-201); maize tb1 promoter(Hubbarda, et al., (2002) Genetics 162:1927-1935 and Wang, et al.,(1999) Nature 398:236-239); maize eep2 promoter (U.S. patent applicationSer. No. 10/817,483); maize thioredoxinH promoter (U.S. ProvisionalPatent Application Ser. No. 60/514,123); maize Zm40 promoter (U.S. Pat.No. 6,403,862 and WO 2001/2178); maize mLIP15 promoter (U.S. Pat. No.6,479,734); maize ESR promoter (U.S. patent application Ser. No.10/786,679); maize PCNA2 promoter (U.S. patent application Ser. No.10/388,359); maize cytokinin oxidase promoters (U.S. patent applicationSer. No. 11/094,917); promoters disclosed in Weigal, et al., (1992) Cell69:843-859; Accession Number AJ131822; Accession Number Z71981;Accession Number AF049870; and shoot-preferred promoters disclosed inMcAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385. Other dividingcell or meristematic tissue-preferred promoters that may be of interesthave been disclosed in Ito, et al., (1994) Plant Mol. Biol. 24:863-878;Regad, et al., (1995) Mo. Gen. Genet. 248:703-711; Shaul, et al., (1996)Proc. Natl. Acad. Sci. 93:4868-4872; Ito, et al., (1997) Plant J.11:983-992; and Trehin, et al., (1997) Plant Mol. Biol. 35:667-672, allof which are hereby incorporated by reference herein.

Inflorescence-preferred promoters include the promoter of chalconesynthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109),LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), pollenspecific genes (Albani, et al., (1990) Plant Mol. Biol. 15:605, Zm13(Buerrero, et al., (1993) Mol. Gen. Genet. 224:161-168), maizepollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol.18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) ThePlant Journal 2:713-721), and B. napus pollen specific genes (Arnoldo,et al., (1992) J. Cell. Biochem, Abstract Number Y101204).

Stress-inducible promoters include salt-inducible orwater-stress-inducible promoters such as P5CS (Zang, et al., (1997)Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a(Hajela, et al., (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm,et al., (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet, et al.,(1998) FEBS Lett. 423-324-328), ci7 (Kirch, et al., (1997) Plant Mol.Biol. 33:897-909), ci21A (Schneider, et al., (1997) Plant Physiol.113:335-45); drought-inducible promoters, such as, Trg-31 (Chaudhary, etal., (1996) Plant Mol. Biol. 30:1247-57); osmotic inducible promoters,such as, Rab17 (Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93;Busk, (1997) Plant J 11(6):1285-1295) and osmotin (Raghothama, et al.,(1993) Plant Mol Biol 23:1117-28) and, heat inducible promoters, suchas, heat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75;Marrs, et al., (1993) Dev. Genet. 14:27-41) and smHSP (Waters, et al.,(1996) J. Experimental Botany 47:325-338). Other stress-induciblepromoters include rip2 (U.S. Pat. No. 5,332,808 and US PatentApplication Publication Number 2003/0217393), and rd29a(Yamaguchi-Shinozaki, et al., (1993) Mol. Gen. Genetics 236:331-340; seealso, GenBank accession D13044). Stress-insensitive promoters can alsobe used in the methods of the invention.

Nitrogen-responsive promoters can also be used in the methods of theinvention. Such promoters include, but are not limited to, the 22 kDaZein promoter (Spena, et al., (1982) EMBO J. 1:1589-1594 and Muller, etal., (1995) J. Plant Physiol 145:606-613); the 19 kDa zein promoter(Pedersen, et al., (1982) Cell 29:1019-1025); the 14 kDa zein promoter(Pedersen, et al., (1986) J. Biol. Chem. 261:6279-6284), the b-32promoter (Lohmer, et al., (1991) EMBO J 10:617-624) and the nitritereductase (NiR) promoter (Rastogi, et al., (1997) Plant Mol Biol.34(3):465-76 and Sander, et al., (1995) Plant Mol Biol. 27(1):165-77).For a review of consensus sequences found in nitrogen-induced promoters,see for example, Muller, et al., (1997) The Plant Journal 12:281-291.

Chemically-regulated promoters can be used to modulate the expression ofa gene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemically-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemically-induciblepromoters are known in the art and include, but are not limited to, themaize In2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237, and U.S.Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Additional inducible promoters include heat shock promoters, such asGmhsp17.5-E (soybean) (Czarnecka, et al., (1989) Mol Cell Biol.9(8):3457-3463); APX1 gene promoter (Arabidopsis) (Storozhenko, et al.,(1998) Plant Physiol. 118(3):1005-1014): Ha hsp17.7 G4 (Helianthusannuus) (Almoguera, et al., (2002) Plant Physiol. 129(1):333-341 andMaize Hsp70 (Rochester, et al., (1986) EMBO J. 5: 451-8).

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868) or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a gene of the presentinvention so as to control the expression of the gene. Gene expressioncan be modulated under conditions suitable for plant growth so as toalter the total concentration and/or alter the composition of thepolypeptides of the present invention in plant cell.

Thus, the present invention provides compositions, and methods formaking, heterologous promoters and/or enhancers operably linked to anative, endogenous (i.e., nonheterologous) form of a polynucleotide ofthe present invention.

Methods for identifying promoters with a particular expression pattern,in terms of, e.g., tissue type, cell type, stage of development and/orenvironmental conditions, are well known in the art. See, e.g., TheMaize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer,N.Y. (1994); Corn and Corn Improvement, 3rd edition, Chapter 6, Spragueand Dudley, Eds., American Society of Agronomy, Madison, Wis. (1988).

A typical step in promoter isolation methods is identification of geneproducts that are expressed with some degree of specificity in thetarget tissue. Amongst the range of methodologies are: differentialhybridization to cDNA libraries; subtractive hybridization; differentialdisplay; differential 2-D protein gel electrophoresis; DNA probe arraysand isolation of proteins known to be expressed with some specificity inthe target tissue. Such methods are well known to those of skill in theart. Commercially available products for identifying promoters are knownin the art such as Clontech's (Palo Alto, Calif.) Universal GenomeWalker Kit.

For the protein-based methods, it is helpful to obtain the amino acidsequence for at least a portion of the identified protein, and then touse the protein sequence as the basis for preparing a nucleic acid thatcan be used as a probe to identify either genomic DNA directly, orpreferably, to identify a cDNA clone from a library prepared from thetarget tissue. Once such a cDNA clone has been identified, that sequencecan be used to identify the sequence at the 5′ end of the transcript ofthe indicated gene. For differential hybridization, subtractivehybridization and differential display, the nucleic acid sequenceidentified as enriched in the target tissue is used to identify thesequence at the 5′ end of the transcript of the indicated gene. Oncesuch sequences are identified, starting either from protein sequences ornucleic acid sequences, any of these sequences identified as being fromthe gene transcript can be used to screen a genomic library preparedfrom the target organism. Methods for identifying and confirming thetranscriptional start site are well known in the art.

In the process of isolating promoters expressed under particularenvironmental conditions or stresses, or in specific tissues, or atparticular developmental stages, a number of genes are identified thatare expressed under the desired circumstances, in the desired tissue orat the desired stage. Further analysis will reveal expression of eachparticular gene in one or more other tissues of the plant. One canidentify a promoter with activity in the desired tissue or condition butthat does not have activity in any other common tissue.

To identify the promoter sequence, the 5′ portions of the clonesdescribed here are analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10bp located approximately 20 to 40 base pairs upstream of thetranscription start site. Identification of the TATA box is well knownin the art. For example, one way to predict the location of this elementis to identify the transcription start site using standard RNA-mappingtechniques such as primer extension, S 1 analysis, and/or RNaseprotection. To confirm the presence of the AT-rich sequence, astructure-function analysis can be performed involving mutagenesis ofthe putative region and quantification of the mutation's effect onexpression of a linked downstream reporter gene. See, e.g., The MaizeHandbook, Chapter 114, Freeling and Walbot, Eds., Springer, N.Y.,(1994).

In plants, further upstream from the TATA box, at positions −80 to −100,there is typically a promoter element (i.e., the CAAT box) with a seriesof adenines surrounding the trinucleotide G (or T) N G. Messing, et al.,in Genetic Engineering in Plants, Kosage, Meredith and Hollaender, Eds.,pp. 221-227 1983. In maize, there is no well conserved CAAT box butthere are several short, conserved protein-binding motifs upstream ofthe TATA box. These include motifs for the trans-acting transcriptionfactors involved in light regulation, anaerobic induction, hormonalregulation or anthocyanin biosynthesis, as appropriate for each gene.

Once promoter and/or gene sequences are known, a region of suitable sizeis selected from the genomic DNA that is 5′ to the transcriptionalstart, or the translational start site, and such sequences are thenlinked to a coding sequence. If the transcriptional start site is usedas the point of fusion, any of a number of possible 5′ untranslatedregions can be used in between the transcriptional start site and thepartial coding sequence. If the translational start site at the 3′ endof the specific promoter is used, then it is linked directly to themethionine start codon of a coding sequence.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes or alternatively from another plantgene or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, (1988)Mol. Cell Biol. 8: 4395-4405; Callis, et al., (1987) Genes Dev.1:1183-1200. Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adhl-S intron 1, 2 and 6, the Bronze-1 intron are known inthe art. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, N.Y. (1994).

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or genetic in resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotic kanamycin, and the ALS gene encodesresistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al.,(1987) Meth. in Enzymol. 153:253-277. These vectors are plantintegrating vectors in that on transformation, the vectors integrate aportion of vector DNA into the genome of the host plant. Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl, et al., (1987) Gene 61:1-11 and Berger, et al., (1989) Proc.Natl. Acad. Sci. USA 86:8402-8406. Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.).

A polynucleotide of the present invention can be expressed in eithersense or antisense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to inhibit gene expression inplants. To accomplish this, a nucleic acid segment from the desired geneis cloned and operably linked to a promoter such that the anti-sensestrand of RNA will be transcribed. The construct is then transformedinto plants and the antisense strand of RNA is produced.

In plant cells, it has been shown that antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theenzyme of interest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad.Sci. USA 85:8805-8809; and Hiatt, et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression. Introduction ofnucleic acid configured in the sense orientation has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to modulate expression ofendogenous genes, see, Napoli, et al., (1990) The Plant Ce112:279-289and U.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff, et al., (1988)Nature 334:585 591. A variety of cross-linking agents, alkylating agentsand radical generating species as pendant groups on polynucleotides ofthe present invention can be used to bind, label, detect and/or cleavenucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan.

The present invention further provides a protein comprising apolypeptide having a specified sequence identity with a polypeptide ofthe present invention. The percentage of sequence identity is an integerselected from the group consisting of from 60 to 99. Exemplary sequenceidentity values include 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%.

As those of skill will appreciate, the present invention includescatalytically active polypeptides of the present invention (i.e.,enzymes). Catalytically active polypeptides have a specific activity ofat least 20%, 30% or 40% and preferably at least 50%, 60% or 70% andmost preferably at least 80%, 90% or 95% that of the native(non-synthetic), endogenous polypeptide. Further, the substratespecificity (kcat/Km) is optionally substantially similar to the native(non-synthetic), endogenous polypeptide. Typically, the Km will be atleast 30%, 40% or 50%, that of the native (non-synthetic), endogenouspolypeptide and more preferably at least 60%, 70%, 80% or 90%. Methodsof assaying and quantifying measures of enzymatic activity and substratespecificity (heat/Km) are well known to those of skill in the art.

Generally, the proteins of the present invention will, when presented asan immunogen, elicit production of an antibody specifically reactive toa polypeptide of the present invention. Further, the proteins of thepresent invention will not bind to antisera raised against a polypeptideof the present invention which has been fully immunosorbed with the samepolypeptide. Immunoassays for determining binding are well known tothose of skill in the art. A preferred immunoassay is a competitiveimmunoassay as discussed, infra. Thus, the proteins of the presentinvention can be employed as immunogens for constructing antibodiesimmunoreactive to a protein of the present invention for such exemplaryutilities as immunoassays or protein purification techniques.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or regulatable), followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression or incorporation of thetargeting molecule into a fusion protein.

Such modifications are well known to those of skill in the art andinclude, for example, a methionine added at the amino terminus toprovide an initiation site or additional amino acids (e.g., poly H is)placed on either terminus to create conveniently located purificationsequences. Restriction sites or termination codons can also beintroduced.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatake,et al., (1981) Nature 292:128). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracyclineor chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene22:229-235; Mosbach, et al., (1983) Nature 302:543-545).

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, etal., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) isa well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeast for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen).

Suitable vectors usually have expression control sequences, such aspromoters, including 3-phosphoglycerate kinase or alcohol oxidase and anorigin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysate. The monitoring of the purification process canbe accomplished by using Western blot techniques or radioimmunoassay orother standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cells. Mammalian cell systems often will be in the form ofmonolayers of cells although mammalian cell suspensions may also beused. A number of suitable host cell lines capable of expressing intactproteins have been developed in the art, and include the HEK293, BHK21and CHO cell lines. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter (e.g., the CMV promoter, a HSVtk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986)Immunol. Rev. 89:49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production ofproteins of the present invention are available, for instance, from theAmerican Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, army worm, moth andDrosophila cell lines such as a Schneider cell line (see, Schneider,(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, Glover, Ed., IRL Press, Arlington, Va. pp.213-238 (1985).

Increasing the Activity and/or Level of an Ethylene Signaling AssociatedPolypeptide

Methods are provided to increase the activity and/or level of theethylene signaling associated polypeptide of the invention. An increasein the level and/or activity of the ethylene signaling associatedpolypeptide of the invention can be achieved by providing to the plantan ethylene signaling associated polypeptide. The ethylene signalingassociated polypeptide can be provided by introducing the amino acidsequence encoding the ethylene signaling associated polypeptide into theplant, introducing into the plant a nucleotide sequence encoding anethylene signaling associated polypeptide or alternatively by modifyinga genomic locus encoding the ethylene signaling associated polypeptideof the invention.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having enhanced activity, such as. It is also recognizedthat the methods of the invention may employ a polynucleotide that isnot capable of directing, in the transformed plant, the expression of aprotein or an RNA. Thus, the level and/or activity of an ethylenesignaling associated polypeptide may be increased by altering the geneencoding the ethylene signaling associated polypeptide or its promoter.See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868. Therefore mutagenized plants that carry mutations inethylene signaling associated genes, where the mutations increaseexpression of the ethylene signaling associated gene or increase theethylene signaling associated activity of the encoded ethylene signalingassociated polypeptide are provided.

Reducing the Activity and/or Level of an Ethylene Signaling AssociatedPolypeptide

Methods are provided to reduce or eliminate the activity of an ethylenesignaling associated polypeptide of the invention by transforming aplant cell with an expression cassette that expresses a polynucleotidethat inhibits the expression of the ethylene signaling associatedpolypeptide. The polynucleotide may inhibit the expression of theethylene signaling associated polypeptide directly, by preventingtranscription or translation of the ethylene signaling associatedmessenger RNA, or indirectly, by encoding a polypeptide that inhibitsthe transcription or translation of an ethylene signaling associatedgene encoding ethylene signaling associated polypeptide. Methods forinhibiting or eliminating the expression of a gene in a plant are wellknown in the art, and any such method may be used in the presentinvention to inhibit the expression of ethylene signaling associatedpolypeptide.

In accordance with the present invention, the expression of an ethylenesignaling associated polypeptide is inhibited if the protein level ofthe ethylene signaling associated polypeptide is less than 70% of theprotein level of the same ethylene signaling associated polypeptide in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that ethylene signaling associated polypeptide. Inparticular embodiments of the invention, the protein level of theethylene signaling associated polypeptide in a modified plant accordingto the invention is less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, less than 5% or less than 2% ofthe protein level of the same ethylene signaling associated polypeptidein a plant that is not a mutant or that has not been geneticallymodified to inhibit the expression of that ethylene signaling associatedpolypeptide. The expression level of the ethylene signaling associatedpolypeptide may be measured directly, for example, by assaying for thelevel of ethylene signaling associated polypeptide expressed in theplant cell or plant, or indirectly, for example, by measuring theethylene response in the plant cell or plant, or by measuring thephenotypic changes in the plant. Methods for performing such assays aredescribed elsewhere herein.

In other embodiments of the invention, the activity of the ethylenesignaling associated polypeptide is reduced or eliminated bytransforming a plant cell with an expression cassette comprising apolynucleotide encoding a polypeptide that inhibits the activity of anethylene signaling associated polypeptide. The activity of an ethylenesignaling associated polypeptide is inhibited according to the presentinvention if the activity of the ethylene signaling associatedpolypeptide is less than 70% of the activity of the same ethylenesignaling associated polypeptide in a plant that has not been modifiedto inhibit the ethylene signaling associated activity of thatpolypeptide. In particular embodiments of the invention, the ethylenesignaling associated activity of the ethylene signaling associatedpolypeptide in a modified plant according to the invention is less than60%, less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10% or less than 5% of the ethylene signaling associated activityof the same polypeptide in a plant that that has not been modified toinhibit the expression of that ethylene signaling associatedpolypeptide. The ethylene signaling associated activity of an ethylenesignaling associated polypeptide is “eliminated” according to theinvention when it is not detectable by the assay methods describedelsewhere herein. Methods of determining the alteration of activity ofan ethylene signaling associated polypeptide are described elsewhereherein.

In other embodiments, the activity of an ethylene signaling associatedpolypeptide may be reduced or eliminated by disrupting the gene encodingthe ethylene signaling associated polypeptide. The invention encompassesmutagenized plants that carry mutations in ethylene signaling associatedgenes, where the mutations reduce expression of the ethylene signalingassociated gene or inhibit the activity of the encoded ethylenesignaling associated polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of anethylene signaling associated polypeptide. In addition, more than onemethod may be used to reduce the activity of a single ethylene signalingassociated polypeptide.

1. Polynucleotide-Based Methods:

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of an ethylene signalingassociated polypeptide of the invention. The term “expression” as usedherein refers to the biosynthesis of a gene product, including thetranscription and/or translation of said gene product. For example, forthe purposes of the present invention, an expression cassette capable ofexpressing a polynucleotide that inhibits the expression of at least oneethylene signaling associated polypeptide is an expression cassettecapable of producing an RNA molecule that inhibits the transcriptionand/or translation of at least one ethylene signaling associatedpolypeptide of the invention. The “expression” or “production” of aprotein or polypeptide from a DNA molecule refers to the transcriptionand translation of the coding sequence to produce the protein orpolypeptide, while the “expression” or “production” of a protein orpolypeptide from an RNA molecule refers to the translation of the RNAcoding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of an ethylenesignaling associated polypeptide are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of anethylene signaling associated polypeptide may be obtained by sensesuppression or cosuppression. For cosuppression, an expression cassetteis designed to express an RNA molecule corresponding to all or part of amessenger RNA encoding an ethylene signaling associated polypeptide inthe “sense” orientation. Over expression of the RNA molecule can resultin reduced expression of the native gene. Accordingly, multiple plantlines transformed with the cosuppression expression cassette arescreened to identify those that show the greatest inhibition of ethylenesignaling associated polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the ethylene signaling associated polypeptide,all or part of the 5′ and/or 3′ untranslated region of an ethylenesignaling associated polypeptide transcript, or all or part of both thecoding sequence and the untranslated regions of a transcript encoding anethylene signaling associated polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for theethylene signaling associated polypeptide, the expression cassette isdesigned to eliminate the start codon of the polynucleotide so that noprotein product will be translated.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001)Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731;Yu, et al., (2003) Phytochemistry 63:753-763; and U.S. Pat. Nos.5,034,323, 5,283,184 and 5,942,657, each of which is herein incorporatedby reference. The efficiency of cosuppression may be increased byincluding a poly-dT region in the expression cassette at a position 3′to the sense sequence and 5′ of the polyadenylation signal. See, USPatent Application Publication Number 2002/0048814, herein incorporatedby reference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323, herein incorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe ethylene signaling associated polypeptide may be obtained byantisense suppression. For antisense suppression, the expressioncassette is designed to express an RNA molecule complementary to all orpart of a messenger RNA encoding the ethylene signaling associatedpolypeptide. Over expression of the antisense RNA molecule can result inreduced expression of the native gene. Accordingly, multiple plant linestransformed with the antisense suppression expression cassette arescreened to identify those that show the greatest inhibition of ethylenesignaling associated polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the ethylenesignaling associated polypeptide, all or part of the complement of the5′ and/or 3′ untranslated region of the ethylene signaling associatedtranscript, or all or part of the complement of both the coding sequenceand the untranslated regions of a transcript encoding the ethylenesignaling associated polypeptide. In addition, the antisensepolynucleotide may be fully complementary (i.e., 100% identical to thecomplement of the target sequence) or partially complementary (i.e.,less than 100% identical to the complement of the target sequence) tothe target sequence. Antisense suppression may be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods forusing antisense suppression to inhibit the expression of endogenousgenes in plants are described, for example, in Liu, et al., (2002) PlantPhysiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657 eachof which is herein incorporated by reference. Efficiency of antisensesuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the antisense sequence and 5′ ofthe polyadenylation signal. See, US Patent Application PublicationNumber 2002/0048814, herein incorporated by reference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of anethylene signaling associated polypeptide may be obtained bydouble-stranded RNA (dsRNA) interference. For dsRNA interference, asense RNA molecule like that described above for cosuppression and anantisense RNA molecule that is fully or partially complementary to thesense RNA molecule are expressed in the same cell, resulting ininhibition of the expression of the corresponding endogenous messengerRNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of ethylene signaling associated polypeptideexpression. Methods for using dsRNA interference to inhibit theexpression of endogenous plant genes are described in Waterhouse, etal., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu, et al.,(2002) Plant Physiol. 129:1732-1743, and WO 1999/49029, WO 1999/53050,WO 1999/61631 and WO 2000/49035, each of which is herein incorporated byreference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression of anethylene signaling associated polypeptide may be obtained by hairpin RNA(hpRNA) interference or intron-containing hairpin RNA (ihpRNA)interference. These methods are highly efficient at inhibiting theexpression of endogenous genes. See, Waterhouse and Helliwell, (2003)Nat. Rev. Genet. 4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Alternatively, thebase-paired stem region may correspond to a portion of a promotersequence controlling expression of the gene to be inhibited. Thus, thebase-paired stem region of the molecule generally determines thespecificity of the RNA interference. hpRNA molecules are highlyefficient at inhibiting the expression of endogenous genes, and the RNAinterference they induce is inherited by subsequent generations ofplants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731 and Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38. Methods for using hpRNA interference to inhibit or silence theexpression of genes are described, for example, in Chuang andMeyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Waterhouseand Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al., BMCBiotechnology 3:7 and US Patent Application Publication Number2003/0175965, each of which is herein incorporated by reference. Atransient assay for the efficiency of hpRNA constructs to silence geneexpression in vivo has been described by Panstruga, et al., (2003) Mol.Biol. Rep. 30:135-140, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295and US Patent Application Publication Number 2003/0180945, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 2002/00904; Mette, et al., (2000)EMBO J 19:5194-5201; Matzke, et al., (2001) Curr. Opin. Genet. Devel.11:221-227; Scheid, et al., (2002) Proc. Natl. Acad. Sci., USA99:13659-13662; Aufsaftz, et al., (2002) Proc. Nat'l. Acad. Sci.99(4):16499-16506; Sijen, et al., Curr. Biol. (2001) 11:436-440), hereinincorporated by reference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for the ethylene signaling associatedpolypeptide). Methods of using amplicons to inhibit the expression ofendogenous plant genes are described, for example, in Angell andBaulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe, (1999)Plant J. 20:357-362 and U.S. Pat. No. 6,646,805, each of which is hereinincorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of the ethylene signaling associatedpolypeptide. Thus, the polynucleotide causes the degradation of theendogenous messenger RNA, resulting in reduced expression of theethylene signaling associated polypeptide. This method is described, forexample, in U.S. Pat. No. 4,987,071, herein incorporated by reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression of anethylene signaling associated polypeptide may be obtained by RNAinterference by expression of a gene encoding a micro RNA (miRNA).miRNAs are regulatory agents consisting of about 22 ribonucleotides.miRNA are highly efficient at inhibiting the expression of endogenousgenes. See, for example Javier, et al., (2003) Nature 425:257-263,herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of ethylene signaling associatedexpression, the 22-nucleotide sequence is selected from an ethylenesignaling associated transcript sequence and contains 22 nucleotides ofsaid ethylene signaling associated sequence in sense orientation and 21nucleotides of a corresponding antisense sequence that is complementaryto the sense sequence. miRNA molecules are highly efficient atinhibiting the expression of endogenous genes, and the RNA interferencethey induce is inherited by subsequent generations of plants.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding an ethylene signaling associated polypeptide,resulting in reduced expression of the gene. In particular embodiments,the zinc finger protein binds to a regulatory region of an ethylenesignaling associated gene. In other embodiments, the zinc finger proteinbinds to a messenger RNA encoding an ethylene signaling associatedpolypeptide and prevents its translation. Methods of selecting sites fortargeting by zinc finger proteins have been described, for example, inU.S. Pat. No. 6,453,242, and methods for using zinc finger proteins toinhibit the expression of genes in plants are described, for example, inUS Patent Application Publication Number 2003/0037355, each of which isherein incorporated by reference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one ethylene signaling associatedpolypeptide, and reduces the enhanced activity of the ethylene signalingassociated polypeptide. In another embodiment, the binding of theantibody results in increased turnover of the antibody-ethylenesignaling associated complex by cellular quality control mechanisms. Theexpression of antibodies in plant cells and the inhibition of molecularpathways by expression and binding of antibodies to proteins in plantcells are well known in the art. See, for example, Conrad and Sonnewald,(2003) Nature Biotech. 21:35-36, incorporated herein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of anethylene signaling associated polypeptide is reduced or eliminated bydisrupting the gene encoding the ethylene signaling associatedpolypeptide. The gene encoding the ethylene signaling associatedpolypeptide may be disrupted by any method known in the art. Forexample, in one embodiment, the gene is disrupted by transposon tagging.In another embodiment, the gene is disrupted by mutagenizing plantsusing random or targeted mutagenesis, and selecting for plants that havereduced activity.

i. Transposon Tagging

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the ethylene signaling activity of one or more ethylenesignaling associated polypeptide. Transposon tagging comprises insertinga transposon within an endogenous ethylene signaling associated gene toreduce or eliminate expression of the ethylene signaling associatedpolypeptide.

In this embodiment, the expression of one or more ethylene signalingassociated polypeptide is reduced or eliminated by inserting atransposon within a regulatory region or coding region of the geneencoding the ethylene signaling associated polypeptide. A transposonthat is within an exon, intron, 5′ or 3′ untranslated sequence, apromoter, or any other regulatory sequence of an ethylene signalingassociated gene may be used to reduce or eliminate the expression and/oractivity of the encoded ethylene signaling associated polypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes, et al., (1999) Trends PlantSci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett.179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al.,(2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol.2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice,et al., (1999) Genetics 153:1919-1928). In addition, the TUSC processfor selecting Mu insertions in selected genes has been described inBensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science274:1537-1540 and U.S. Pat. No. 5,962,764, each of which is hereinincorporated by reference.

ii. Mutant Plants with Reduced Activity

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see, Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics154:421-436, each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See, McCallum,et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction (enhanced activity) of the encoded protein are well known inthe art. Insertional mutations in gene exons usually result innull-mutants. Mutations in conserved residues are particularly effectivein inhibiting the activity of the encoded protein. Conserved residues ofplant ethylene signaling associated polypeptides suitable formutagenesis with the goal to eliminate ethylene signaling associatedactivity have been described. Such mutants can be isolated according towell-known procedures, and mutations in different ethylene signalingassociated loci can be stacked by genetic crossing. See, for example,Gruis, et al., (2002) Plant Cell 14:2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba, et al., (2003) PlantCell 15:1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more ethylene signaling associated polypeptide.Examples of other methods for altering or mutating a genomic nucleotidesequence in a plant are known in the art and include, but are notlimited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors,RNA:DNA repair vectors, mixed-duplex oligonucleotides,self-complementary RNA:DNA oligonucleotides and recombinogenicoligonucleobases. Such vectors and methods of use are known in the art.See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325;5,760,012; 5,795,972 and 5,871,984, each of which are hereinincorporated by reference. See also, WO 1998/49350, WO 1999/07865, WO1999/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA96:8774-8778, each of which is herein incorporated by reference.

Transfection/Transformation of Cells

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied.

Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for effectivetransformation/transfection may be employed.

A. Plant Transformation

A DNA sequence coding for the desired polypeptide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength protein, will be used to construct a recombinant expressioncassette which can be introduced into the desired plant.

Isolated nucleic acid acids of the present invention can be introducedinto plants according to techniques known in the art. Generally,recombinant expression cassettes as described above and suitable fortransformation of plant cells are prepared. Techniques for transforminga wide variety of higher plant species are well known and described inthe technical, scientific, and patent literature. See, for example,Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. For example, theDNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation, polyethylene glycol(PEG), poration, particle bombardment, silicon fiber delivery, ormicroinjection of plant cell protoplasts or embryogenic callus. See,e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp. 197213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods. eds. Gamborg and Phillips, Springer-VerlagBerlin Heidelberg New York, 1995. Alternatively, the DNA constructs maybe combined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.

The introduction of DNA constructs using PEG precipitation is describedin Paszkowski, et al., (1984) Embo J. 3:2717-2722. Electroporationtechniques are described in Fromm, et al., (1985) Proc. Natl. Acad. Sci.(USA) 82:5824. Ballistic transformation techniques are described inKlein, et al., (1987) Nature 327:70-73.

Agrobacterium tumefaciens-mediated transformation techniques are welldescribed in the scientific literature. See, for example Horsch, et al.,(1984) Science 233:496-498, and Fraley, et al., (1983) Proc. Natl. Acad.Sci. (USA) 80:4803. Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, Rigby, Ed.,London, Academic Press, 1987 and Lichtenstein and Draper, In: DNACloning, Vol. II, Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 1988/02405 published Apr. 7, 1988) describes the useof A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 orpARC16; (2) liposome-mediated DNA uptake (see, e.g.,Freeman, et al., (1984) Plant Cell Physiol. 25:1353); (3) the vortexingmethod (see, e.g., Kindle, (1990) Proc. Natl. Acad. Sci. (USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou, et al., (1983) Methods in Enzymology101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena, et al., (1987) Nature 325:274.

DNA can also be injected directly into the cells of immature embryos andthe rehydration of desiccated embryos as described by Neuhaus, et al.,(1987) Theor. Appl. Genet. 75:30 and Benbrook, et al., (1986) inProceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54. Avariety of plant viruses that can be employed as vectors are known inthe art and include cauliflower mosaic virus (CaMV), geminivirus, bromemosaic virus and tobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. (1977).

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., (1963) J. Am. Chem. Soc.85:2149-2156 and Stewart, et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art and include,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

Transgenic Plant Regeneration

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. For transformation and regeneration of maize, see,Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans, et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horsch,et al., (1985) Science 227:1229-1231. In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley, et al., (1983) Proc. Natl. Acad.Sci. USA 80:4803. This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Kleen, et al., (1987) Ann. Rev. of Plant Phys. 38:467-486. Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, Weissbach and Weissbach, eds., Academic Press, Inc.,San Diego, Calif. (1988). This regeneration and growth process includesthe steps of selection of transformant cells and shoots, rooting thetransformant shoots and growth of the plantlets in soil. For maize cellculture and regeneration see generally, The Maize Handbook, Freeling andWalbot, Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3rdedition, Sprague and Dudley Eds., American Society of Agronomy, Madison,Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.

Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences. Transgenic plants expressing the selectable marker can bescreened for transmission of the nucleic acid of the present inventionby, for example, standard immunoblot and DNA detection techniques.Transgenic lines are also typically evaluated on levels of expression ofthe heterologous nucleic acid. Expression at the RNA level can bedetermined initially to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includePCR amplification assays using oligonucleotide primers designed toamplify only the heterologous RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. The RNA-positiveplants can then analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, nontransgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Modulation of Polypeptide Levels and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or ratio of the polypeptidesof the present invention in a plant or part thereof. Modulation can beeffected by increasing or decreasing the concentration and/or the ratioof the polypeptides of the present invention in a plant.

The method comprises introducing into a plant cell a recombinantexpression cassette comprising a polynucleotide of the present inventionas described above to obtain a transformed plant cell, culturing thetransformed plant cell under plant cell growing conditions and inducingor repressing expression of a polynucleotide of the present invention inthe plant for a time sufficient to modulate concentration and/or theratios of the polypeptides in the plant or plant part.

In some embodiments, the concentration and/or ratios of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native genes ofthe present invention can be altered via substitution, addition,insertion, or deletion to decrease activity of the encoded enzyme. See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.And in some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.

Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or ratios of polypeptides of the present invention inthe plant. Plant forming conditions are well known in the art anddiscussed briefly, supra.

In general, concentration or the ratios of the polypeptides is increasedor decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% relative to a native control plant, plant part or cell lacking theaforementioned recombinant expression cassette. Modulation in thepresent invention may occur during and/or subsequent to growth of theplant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

Molecular Markers

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Optionally, the plant is amonocot, such as maize or sorghum. Genotyping provides a means ofdistinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Clark, Ed., Plant Molecular Biology: A Laboratory Manual. Berlin,Springer Verlag, 1997. Chapter 7. For molecular marker methods, seegenerally, “The DNA Revolution” in: Paterson, Genome Mapping in Plants(Austin, Tex., Academic Press/R. G. Landis Company, 1996) pp. 7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentsresulting from nucleotide sequence variability. As is well known tothose of skill in the art, RFLPs are typically detected by extraction ofgenomic DNA and digestion with a restriction enzyme. Generally, theresulting fragments are separated according to size and hybridized witha probe; single copy probes are preferred. Restriction fragments fromhomologous chromosomes are revealed.

Differences in fragment size among alleles represent an RFLP. Thus, thepresent invention further provides a means to follow segregation of agene or nucleic acid of the present invention as well as chromosomalsequences genetically linked to these genes or nucleic acids using suchtechniques as RFLP analysis. Linked chromosomal sequences are within 50centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10cM, more preferably within 5, 3, 2 or 1 cM of a gene of the presentinvention.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes selectively hybridize, underselective hybridization conditions, to a gene encoding a polynucleotideof the present invention. In preferred embodiments, the probes areselected from polynucleotides of the present invention.

Typically, these probes are cDNA probes or restriction-enzyme treated(e.g., Pst I) genomic clones. The length of the probes is discussed ingreater detail, supra, but are typically at least 15 bases in length,more preferably at least 20, 25, 30, 35, 40 or 50 bases in length.Generally, however, the probes are less than about 1 kilobase in length.Preferably, the probes are single copy probes that hybridize to a uniquelocus in a haploid chromosome complement. Some exemplary restrictionenzymes employed in RFLP mapping are EcoRI, EcoRv and SstI. As usedherein the term “restriction enzyme” includes reference to a compositionthat recognizes and, alone or in conjunction with another composition,cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present of said genomic DNA; (c)detecting therefrom a RFLP. Other methods of differentiating polymorphic(allelic) variants of polynucleotides of the present invention can behad by utilizing molecular marker techniques well known to those ofskill in the art including such techniques as: 1) single strandedconformation analysis (SSCA); 2) denaturing gradient gel electrophoresis(DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides(ASOs); 5) the use of proteins which recognize nucleotide mismatches,such as the E. coli mutS protein and 6) allele-specific PCR. Otherapproaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage(CMC). Thus, the present invention further provides a method ofgenotyping comprising the steps of contacting, under stringenthybridization conditions, a sample suspected of comprising apolynucleotide of the present invention with a nucleic acid probe.Generally, the sample is a plant sample; preferably, a sample suspectedof comprising a maize polynucleotide of the present invention (e.g.,gene, mRNA). The nucleic acid probe selectively hybridizes, understringent conditions, to a subsequence of a polynucleotide of thepresent invention comprising a polymorphic marker. Selectivehybridization of the nucleic acid probe to the polymorphic markernucleic acid sequence yields a hybridization complex. Detection of thehybridization complex indicates the presence of that polymorphic markerin the sample. In preferred embodiments, the nucleic acid probecomprises a polynucleotide of the present invention.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 7-methylguanosine cap structure (Drummond, et al.,(1985) Nucleic Acids Res. 13:7375). Negative elements include stableintramolecular 5′UTR stem-loop structures (Muesing, et al., (1987) Cell48:691) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol.and Cell. Biol. 8:284). Accordingly, the present invention provides 5′and/or 3′ untranslated regions for modulation of translation ofheterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT Publication Number WO1997/20078. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509. Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system, and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element, orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be adecreased Km and/or increased KCat over the wild-type protein asprovided herein. In other embodiments, a protein or polynucleotidegenerated from sequence shuffling will have a ligand binding affinitygreater than the non-shuffled wild-type polynucleotide.

The increase in such properties can be at least 110%, 120%, 130%, 140%or at least 150% of the wild-type value.

Generic and Consensus Sequences

Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present inventionand (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice or sorghum.

Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 25, 30 or 40 amino acids in length, or 20, 30, 40, 50, 100or 150 nucleotides in length. As those of skill in the art are aware, aconservative amino acid substitution can be used for amino acids whichdiffer amongst aligned sequence but are from the same conservativesubstitution group as discussed above. Optionally, no more than 1 or 2conservative amino acids are substituted for each 10 amino acid lengthof consensus sequence.

Similar sequences used for generation of a consensus or generic sequenceinclude any number and combination of allelic variants of the same gene,orthologous or paralogous sequences as provided herein. Optionally,similar sequences used in generating a consensus or generic sequence areidentified using the BLAST algorithm's smallest sum probability (P (N)).Various suppliers of sequence-analysis software are listed in chapter 7of Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (Supplement 30).

A polynucleotide sequence is considered similar to a reference sequenceif the smallest sum probability in a comparison of the test nucleic acidto the reference nucleic acid is less than about 0.1, more preferablyless than about 0.01 or 0.001 and most preferably less than about 0.0001or 0.00001. Similar polynucleotides can be aligned and a consensus orgeneric sequence generated using multiple sequence alignment softwareavailable from a number of commercial suppliers such as the GeneticsComputer Group's (Madison, Wis.) PILEUP software, Vector NTI's (NorthBethesda, Md.) ALIGNX or Genecode's (Ann Arbor, Mich.) SEQUENCHER.Conveniently, default parameters of such software can be used togenerate consensus or generic sequences.

Machine Applications

The present invention provides machines, articles of manufacture, andprocesses for identifying, modeling or analyzing the polynucleotides andpolypeptides of the present invention. Identification methods permitidentification of homologues of the polynucleotides or polypeptides ofthe present invention while modeling and analysis methods permitrecognition of structural or functional features of interest.

A. Machines: Data Processing Systems

In one embodiment, the present invention provides a machine having: 1) amemory comprising data representing at least one genetic sequence, 2) agenetic identification, analysis, or modeling program with access to thedata, 3) a data processor which executes instructions according to theprogram using the genetic sequence or a subsequence thereof and 4) anoutput for storing or displaying the results of the data processing.

The machine of the present invention is a data processing system,typically a digital computer. The term “computer” includes one orseveral desktop or portable computers, computer workstations, servers(including intranet or internet servers), mainframes and any integratedsystem comprising any of the above irrespective of whether theprocessing, memory, input, or output of the computer is remote or local,as well as any networking interconnecting the modules of the computer.Data processing can thus be remote or distributed amongst severalprocessors at one or multiple sites. The data processing systemcomprises a data processor, such as a central processing unit (CPU),which executes instructions according to an application program. As usedherein, machines, articles of manufacture and processes are exclusive ofthe machines, manufactures and processes employed by the United StatesPatent and Trademark Office or the European Patent Office when datarepresenting the sequence of a polypeptide or polynucleotide of thepresent invention is used for patentability searches.

The machine of the present invention includes a memory comprising datarepresenting at least one genetic sequence. As used herein, “geneticsequence” refers to the primary sequence (i.e., amino acid or nucleotidesequence) of a polynucleotide or polypeptide of the present invention.The genetic sequence can represent a partial sequence from a full-lengthprotein, genomic DNA or full-length cDNA/mRNA. Nucleic acids or proteinscomprising a genetic sequence that is identified, analyzed or modeledaccording to the present invention can be cloned or synthesized.

As those of skill in the art will be aware, the form of memory of amachine of the present invention, or the particular embodiment of thecomputer readable medium, are not critical elements of the invention andcan take a variety of forms. The memory of such a machine includes, butis not limited to, ROM or RAM or computer readable media such as, butnot limited to, magnetic media such as computer disks or hard drives ormedia such as CD-ROMs, DVDs and the like. The memory comprising the datarepresenting the genetic sequence includes main memory, a register and acache. In some embodiments the data processing system stores the datarepresenting the genetic sequence in memory while processing the dataand wherein successive portions of the data are copied sequentially intoat least one register of the data processor for processing. Thus, thegenetic sequence stored in memory can be a genetic sequence createdduring computer runtime or stored beforehand. The machine of the presentinvention includes a genetic identification, analysis, or modelingprogram (discussed below) with access to the data representing thegenetic sequence. The program can be implemented in software orhardware.

The present invention further contemplates that the machine of thepresent invention will reference, directly or indirectly, a utility orfunction for the polynucleotide or polypeptide of the present invention.For example, the utility/function can be directly referenced as a dataelement in the machine and accessible by the program. Alternatively, theutility/function of the genetic can be indirectly referenced to anelectronic or written record. The function or utility of the geneticsequence can be a function or utility for the genetic sequence or thedata representing the sequence (i.e., the genetic sequence data).

Exemplary function or utilities for the genetic sequence include: 1) itsname (per International Union of Biochemistry and Molecular Biologyrules of nomenclature) or the function of the enzyme or proteinrepresented by the genetic sequence, 2) the metabolic pathway that theprotein represented by the genetic sequence participates in, 3) thesubstrate or product or structural role of the protein represented bythe genetic sequence or 4) the phenotype (e.g., an agronomic orpharmacological trait) affected by modulating expression or activity ofthe protein represented by the genetic sequence.

The machine of the present invention also includes an output fordisplaying, printing or recording the results of the identification,analysis or modeling performed using a genetic sequence of the presentinvention. Exemplary outputs include monitors, printers or variouselectronic storage mechanisms (e.g., floppy disks, hard drives, mainmemory) which can be used to display the results or employed as a meansto input the stored data into a subsequent application or device.

In some embodiments, data representing a genetic sequence of the presentinvention is a data element within a data structure. The data structuremay be defined by the computer programs that define the processes ofidentification, modeling, or analysis (see below) or it may be definedby the programming of separate data storage and retrieval programssubroutines or systems. Thus, the present invention provides a memoryfor storing a data structure that can be accessed by a computerprogrammed to implement a process for identification, analysis, ormodeling of a genetic sequence. The data structure, stored withinmemory, is associated with the data representing the genetic sequenceand reflects the underlying organization and structure of the geneticsequence to facilitate program access to data elements corresponding tological sub-components of the genetic sequence. The data structureenables the genetic sequence to be identified, analyzed or modeled. Theunderlying order and structure of a genetic sequence is datarepresenting the higher order organization of the primary sequence. Suchhigher order structures affect transcription, translation, enzymekinetics or reflects structural domains or motifs.

Exemplary logical sub-components which constitute the higher orderorganization of the genetic sequence include but are not limited to:restriction enzyme sites, endopeptidase sites, major grooves, minorgrooves, beta-sheets, alpha helices, open reading frames (ORFs), 5′untranslated regions (UTRs), 3′ UTRs, ribosome binding sites,glycosylation sites, signal peptide domains, intron-exon junctions,poly-A tails, transcription initiation sites, translation start sites,translation termination sites, methylation sites, zinc finger domains,modified amino acid sites, preproprotein-proprotein junctions,proprotein-protein junctions, transit peptide domains, single nucleotidepolymorphisms (SNPs), simple sequence repeats (SSRs), restrictionfragment length polymorphisms (RFLPs), insertion elements, transmembranespanning regions and stem-loop structures.

In another embodiment, the present invention provides a data processingsystem comprising at least one data structure in memory where the datastructure supports the accession of data representing a genetic sequenceof the present invention. The system also comprises at least one geneticidentification, analysis or modeling program which directs the executionof instructions by the system using the genetic sequence data toidentify, analyze or model at least one data element which is a logicalsub-component of the genetic sequence. An output for the processingresults is also provided.

B. Articles of Manufacture: Computer Readable Media

In one embodiment, the present invention provides a data structure in acomputer readable medium that contains data representing a geneticsequence of the present invention. The data structure is organized toreflect the logical structuring of the genetic sequence, so that thesequence can be analyzed by software programs capable of accessing thedata structure. In particular, the data structures of the presentinvention organize the genetic sequences of the present invention in amanner which allows software tools to perform an identification,analysis or modeling using logical elements of each genetic sequence.

In a further embodiment, the present invention provides amachine-readable media containing a computer program and geneticsequence data. The program provides instructions sufficient to implementa process for effecting the identification, analysis or modeling of thegenetic sequence data. The media also includes a data structurereflecting the underlying organization and structure of the data tofacilitate program access to data elements corresponding to logicalsub-components of the genetic sequence, the data structure beinginherent in the program and in the way in which the program organizesand accesses the data.

An example of a data structure resembles a layered hash table, where inone dimension the base content of the sequence is represented by astring of elements A, T, C, G and N. The direction from the 5′ end tothe 3′ end is reflected by the order from the position 0 to the positionof the length of the string minus one. Such a string, corresponding to anucleotide sequence of interest, has a certain number of substrings,each of which is delimited by the string position of its 5′ end and thestring position of its 3′ end within the parent string. In a seconddimension, each substring is associated with or pointed to one ormultiple attribute fields. Such attribute fields contain annotations tothe region on the nucleotide sequence represented by the substring.

For example, a sequence under investigation is 520 bases long andrepresented by a string named SeqTarget. There is a minor groove in the5′ upstream non-coding region from position 12 to 38, which isidentified as a binding site for an enhancer protein HM-A, which in turnwill increase the transcription of the gene represented by SeqTarget.Here, the substring is represented as (12, 38) and has the followingattributes: [upstream uncoded], [minor groove], [HM-A binding] and[increase transcription upon binding by HM-A]. Similarly, other types ofinformation can be stored and structured in this manner, such asinformation related to the whole sequence, e.g., whether the sequence isa full length viral gene, a mammalian house keeping gene or an EST fromclone X, information related to the 3′ down stream non-coding region,e.g., hair pin structure and information related to various domains ofthe coding region, e.g., Zinc finger.

This data structure is an open structure and is robust enough toaccommodate newly generated data and acquired knowledge. Such astructure is also a flexible structure. It can be trimmed down to a1-Dstring to facilitate data mining and analysis steps, such as clustering,repeat-masking, and HMM analysis. Meanwhile, such a data structure alsocan extend the associated attributes into multiple dimensions. Pointerscan be established among the dimensioned attributes when needed tofacilitate data management and processing in a comprehensive genomicsknowledge base. Furthermore, such a data structure is object-oriented.Polymorphism can be represented by a family or class of sequenceobjects, each of which has an internal structure as discussed above. Thecommon traits are abstracted and assigned to the parent object, whereaseach child object represents a specific variant of the family or class.Such a data structure allows data to be efficiently retrieved, updatedand integrated by the software applications associated with the sequencedatabase and/or knowledge base.

C. Processes: Identification, Analysis or Modeling

The present invention also provides a process of identifying, analyzingor modeling data representing a genetic sequence of the presentinvention. The process comprises: 1) providing a machine having ahardware or software implemented genetic sequence identification,modeling or analysis program with data representing a genetic sequence,2) executing the program while granting it access to the geneticsequence data, and 3) displaying or outputting the results of theidentification, analysis, or modeling. Data structures made by theprocesses of the present invention and embodied within a computerreadable medium are also provided herein.

A further process of the present invention comprises providing a memoryembodied with data representing a genetic sequence and developing withinthe memory a data structure associated with the data and reflecting theunderlying organization and structure of the data to facilitate programaccess to data elements corresponding to logical subcomponents of thesequence. A computer is programmed with a program containinginstructions sufficient to implement the process for effecting theidentification, analysis or modeling of the genetic sequence and theprogram is executed on the computer while granting the program access tothe data and to the data structure within the memory. The programresults are outputted.

Identification, analysis and modeling programs are well known in the artand available commercially. The program typically has at least oneapplication to: 1) identify the structural role or enzymatic function ofthe gene which the genetic sequence encodes or is translated from, 2)analyzes and identifies higher order structures within the geneticsequence or 3) model the physico-chemical properties of a geneticsequence of the present invention in a particular environment.

Included amongst the modeling/analysis tools are methods to: 1)recognize overlapping sequences (e.g., from a sequencing project) with apolynucleotide of the present invention and create an alignment called a“contig”, 2) identify restriction enzyme sites of a polynucleotide ofthe present invention, 3) identify the products of a TI ribonucleasedigestion of a polynucleotide of the present invention, 4) identify PCRprimers with minimal self-complementarity, 5) compute pairwise distancesbetween sequences in an alignment, reconstruct phylogentic trees usingdistance methods and calculate the degree of divergence of two proteincoding regions, 6) identify patterns such as coding regions,terminators, repeats and other consensus patterns in polynucleotides ofthe present invention, 7) identify RNA secondary structure, 8) identifysequence motifs, isoelectric point, secondary structure, hydrophobicityand antigenicity in polypeptides of the present invention, 9) translatepolynucleotides of the present invention and backtranslate polypeptidesof the present invention and 10) compare two protein or nucleic acidsequences and identifying points of similarity or dissimilarity betweenthem.

Identification of the function/utility of a genetic sequence istypically achieved by comparative analysis to a gene/protein databaseand establishing the genetic sequence as a candidate homologue (i.e.,ortholog or paralog) of a gene/protein of known function/utility.

A candidate homologue has statistically significant probability ofhaving the same biological function (e.g., catalyzes the same reaction,binds to homologous proteins/nucleic acids, has a similar structuralrole) as the reference sequence to which it is compared. Sequenceidentity/similarity is frequently employed as a criterion to identifycandidate homologues. In the same vein, genetic sequences of the presentinvention have utility in identifying homologs in animals or other plantspecies, particularly those in the family Gramineae such as, but notlimited to, sorghum, wheat or rice. Function is frequently establishedon the basis of sequence identity/similarity. Exemplary sequencecomparison systems are provided for in sequence analysis software suchas those provided by the Genetics Computer Group (Madison, Wis.) orInforMax</RTI.

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a gene of the present invention or portion thereof can beamplified prior to the step of contacting the nucleic acid sample with apolynucleotide of the present invention. The nucleic acid sample iscontacted with the polynucleotide to form a hybridization complex. Thepolynucleotide hybridizes under stringent conditions to a gene encodinga polypeptide of the present invention. Formation of the hybridizationcomplex is used to detect a gene encoding a polypeptide of the presentinvention in the nucleic acid sample. Those of skill will appreciatethat an isolated nucleic acid comprising a polynucleotide of the presentinvention should lack cross-hybridizing sequences in common withnon-target genes that would yield a false positive result.

Detection of the hybridization complex can be achieved using any numberof well known methods. For example, the nucleic acid sample, or aportion thereof, may be assayed by hybridization formats including butnot limited to, solution phase, solid phase, mixed phase or in situhybridization assays. Briefly, in solution (or liquid) phasehybridizations, both the target nucleic acid and the probe or primer arefree to interact in the reaction mixture. In solid phase hybridizationassays, probes or primers are typically linked to a solid support wherethey are available for hybridization with target nucleic in solution. Inmixed phase, nucleic acid intermediates in solution hybridize to targetnucleic acids in solution as well as to a nucleic acid linked to a solidsupport. In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the various hybridization assay formats: Singer,et al., (1986) Biotechniques 4(3):230-250; Haase, et al., (1984) Methodsin Virology 7:189-226; Wilkinson, The theory and practice of in situhybridization in: In situ Hybridization, Wilkinson, Ed., IRL Press,Oxford University Press, Oxford; and Nucleic Acid Hybridization: APractical Approach, Hames, and Higgins, Eds., IRL Press (1987).

Nucleic Acid Labels and Detection Methods

The means by which nucleic acids of the present invention are labeled isnot a critical aspect of the present invention and can be accomplishedby any number of methods currently known or later developed. Detectablelabels suitable for use in the present invention include any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means.

Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein and thelike), radiolabels (e.g., 3H, 125I, 35S, I4C or 32P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

Nucleic acids of the present invention can be labeled by any one ofseveral methods typically used to detect the presence of hybridizednucleic acids. One common method of detection is the use ofautoradiography using probes labeled with 3H, I25I, 35S, I4C or 32P orthe like. The choice of radioactive isotope depends on researchpreferences due to ease of synthesis, stability and half lives of theselected isotopes. Other labels include ligands which bind to antibodieslabeled with fluorophores, chemiluminescent agents and enzymes.Alternatively, probes can be conjugated directly with labels such asfluorophores, chemiluminescent agents or enzymes. The choice of labeldepends on sensitivity required, ease of conjugation with the probe,stability requirements and available instrumentation. Labeling thenucleic acids of the present invention is readily achieved such as bythe use of labeled PCR primers.

In some embodiments, the label is simultaneously incorporated during theamplification step in the preparation of the nucleic acids. Thus, forexample, polymerase chain reaction (PCR) with labeled primers or labelednucleotides will provide a labeled amplification product. In anotherembodiment, transcription amplification using a labeled nucleotide(e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into thetranscribed nucleic acids.

Non-radioactive probes are often labeled by indirect means. For example,a ligand molecule is covalently bound to the probe. The ligand thenbinds to an anti-ligand molecule which is either inherently detectableor covalently bound to a detectable signal system, such as an enzyme, afluorophore or a chemiluminescent compound. Enzymes of interest aslabels will primarily be hydrolases, such as phosphatases, esterases andglycosidases or oxidoreductases, particularly peroxidases. Fluorescentcompounds include fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Chemiluminescers includeluciferin and 2,3-dihydrophthalazinediones, e.g., luminol.

Ligands and anti-ligands may be varied widely. Where a ligand has anatural anti-ligand, namely ligands such as biotin, thyroxine andcortisol, it can be used in conjunction with its labeled, naturallyoccurring anti-ligands. Alternatively, any haptenic or antigeniccompound can be used in combination with an antibody. Probes can also belabeled by direct conjugation with a label. For example, cloned DNAprobes have been coupled directly to horseradish peroxidase or alkalinephosphatase.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate and colorimetric labels are detected by simply visualizingthe colored label.

Antibodies to Proteins

Antibodies can be raised to a protein of the present invention,including individual, allelic, strain or species variants and fragmentsthereof, both in their naturally occurring (full-length) forms and inrecombinant forms. Additionally, antibodies are raised to these proteinsin either their native configurations or in non-native configurations.Many methods of making antibodies are known to persons of skill. Avariety of analytic methods are available to generate a hydrophilicityprofile of a protein of the present invention. Such methods can be usedto guide the artisan in the selection of peptides of the presentinvention for use in the generation or selection of antibodies which arespecifically reactive, under immunogenic conditions, to a protein of thepresent invention. See, e.g., Janin, (1979) Nature 277:491-492;Wolfenden, et al., (1981) Biochemistry 20:849-855; Kyte and Doolite,(1982) J. Mol. Biol. 157:105-132; Rose, et al., (1985) Science229:834-838. The following discussion is presented as a general overviewof the techniques available; however, one of skill will recognize thatmany variations upon the following methods are known.

A number of immunogens are used to produce antibodies specificallyreactive with a protein of the present invention. An isolatedrecombinant, synthetic, or native polynucleotide of the presentinvention are the preferred antigens for the production of monoclonal orpolyclonal antibodies. Polypeptides of the present invention areoptionally denatured, and optionally reduced, prior to formation ofantibodies for screening expression libraries or other assays in which aputative protein of the present invention is expressed or denatured in anon-native secondary, tertiary or quaternary structure.

The protein of the present invention is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the protein of the presentinvention. Methods of producing polyclonal antibodies are known to thoseof skill in the art. In brief, an antigen, preferably a purifiedprotein, a protein coupled to an appropriate carrier (e.g., GST, keyholelimpet hemanocyanin, etc.) or a protein incorporated into animmunization vector such as a recombinant vaccinia virus (see, U.S. Pat.No. 4,722,848) is mixed with an adjuvant and animals are immunized withthe mixture. The animal's immune response to the immunogen preparationis monitored by taking test bleeds and determining the titer ofreactivity to the protein of interest. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the protein is performed wheredesired (See, e.g., Coligan, (1991) Current Protocols in Immunology,Wiley/Greene, N.Y.; and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Press, NY (1989)).

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of a protein of thepresent invention are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a protein of at least about 5amino acids, more typically the protein is 10 amino acids in length,preferably, 15 amino acids in length and more preferably the protein is20 amino acids in length or greater. The peptides are typically coupledto a carrier protein (e.g., as a fusion protein), or are recombinantlyexpressed in an immunization vector. Antigenic determinants on peptidesto which antibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from hybrid cells secreting thedesired antibody. Monoclonals antibodies are screened for binding to aprotein from which the antigen was derived. Specific monoclonal andpolyclonal antibodies will usually have an antibody binding site with anaffinity constant for its cognate monovalent antigen at least between106-107, usually at least 108, preferably at least 109, more preferablyat least 110 and most preferably at least 111 liters/mole.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Basic and Clinical Immunology, 4th ed., Stites, et al.,Eds., Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane, supra; Goding, Monoclonal Antibodies:Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986);and Kohler and Milstein, (1975) Nature 256:495-497. Summarized briefly,this method proceeds by injecting an animal with an antigen comprising aprotein of the present invention. The animal is then sacrificed andcells taken from its spleen, which are fused with myeloma cells. Theresult is a hybrid cell or “hybridoma” that is capable of reproducing invitro.

The population of hybridomas is then screened to isolate individualclones, each of which secrete a single antibody species to the antigen.In this manner, the individual antibody species obtained are theproducts of immortalized and cloned single B cells from the immuneanimal generated in response to a specific site recognized on theantigenic substance.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse, et al., (1989)Science 246:1275-1281; and Ward, et al., (1989) Nature 341:544-546 andVaughan, et al., (1996) Nature Biotechnology 14:309-314). Alternatively,high avidity human monoclonal antibodies can be obtained from transgenicmice comprising fragments of the unrearranged human heavy and lightchain Ig loci (i.e., mini locus transgenic mice). Fishwild, et al.,(1996) Nature Biotech. 14:845-851. Also, recombinant immunoglobulins maybe produced. See, Cabilly, U.S. Pat. No. 4,816,567 and Queen, et al.,(1989) Proc. Natl. Acad. Sci. 86:10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating proteins of the present invention. Columnsare prepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, SEPHADEX, or the like, where a cell lysateis passed through the column, washed and treated with increasingconcentrations of a mild denaturant, whereby purified protein arereleased.

The antibodies can be used to screen expression libraries for particularexpression products such as normal or abnormal protein. Usually theantibodies in such a procedure are labeled with a moiety allowing easydetection of presence of antigen by antibody binding. Antibodies raisedagainst a protein of the present invention can also be used to raiseanti-idiotypic antibodies. These are useful for detecting or diagnosingvarious pathological conditions related to the presence of therespective antigens.

Frequently, the proteins and antibodies of the present invention will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles andthe like.

Plants exhibiting an altered ethylene-dependent phenotype as comparedwith wild-type plants can be selected among other methods, by visualobservation. For example, an altered ethylene-dependent phenotype may bedetected by utilization of the “triple response.” The “triple response”consists of three distinct morphological changes in dark-grown seedlingsupon exposure to ethylene: inhibition of hypocotyl and root elongation,radial swelling of the stem and exaggeration of the apical hook. Thus, atriple response displayed in the presence of ethylene inhibitors wouldindicate one type of altered ethylene-dependent phenotype. Ethyleneaffects a vast array of agriculturally important plant processes,including fruit ripening, flower and leaf senescence and leafabscission. The ability to control the sensitivity of plants to ethylenecould thus significantly improve the quality and longevity of manycrops. The invention includes plants produced by the method of theinvention, as well as plant tissue and seeds.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Example 1

This example describes the construction of the cDNA libraries. Total RNAfor SEQ ID NO: 1 (EIN3), SEQ ID NO: 3 (EBF1), SEQ ID NO: 5 (EBF2), SEQID NO: 7 (EIN5) or SEQ ID NO: 9 (ERF3) was obtained from maize genotypeHill (Armstrong and Phillips, (1988) Crop Sci. 28:363-369) and forZmEIN3-2 (SEQ ID NO: 1), from night harvested leaf tissue at the V8-V10stage of maize genotype B75. The total RNA was isolated from the maizetissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.)using a modification of the guanidine isothiocyanate/acid-phenolprocedure described by Chomczynski and Sacchi (Chomczynski and Sacchi,(1987) Anal. Biochem. 162:156). In brief, plant tissue samples werepulverized in liquid nitrogen before the addition of the TRIzol Reagent,and then were further homogenized with a mortar and pestle. Addition ofchloroform followed by centrifugation was conducted for separation of anaqueous phase and an organic phase. The total RNA was recovered byprecipitation with isopropyl alcohol from the aqueous phase.

Poly (A)+RNA Isolation

The selection of poly (A)+RNA from total RNA was performed usingPolyATact system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo (dT) primers were used to hybridize to the 3′ poly(A) tails on mRNA. The hybrids were captured using streptavidin coupledto paramagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringency conditions and eluted by RNase-free deionizedwater. cDNA Library Construction cDNA synthesis was performed andunidirectional cDNA libraries were constructed using the SuperScriptPlasmid System (Life Technology Inc. Gaithersburg, Md.). The firststrand of cDNA was synthesized by priming an oligo (dT) primercontaining a Not I site.

The reaction was catalyzed by SuperScript Reverse Transcriptase II at45° C. The second strand of cDNA was labeled withalpha-32P-dCTP and aportion of the reaction was analyzed by agarose gel electrophoresis todetermine cDNA sizes. cDNA molecules smaller than 500 base pairs andunligated adapters were removed by Sephacryl-5400 chromatography. Theselected cDNA molecules were ligated into pSPORTI vector in between ofNot I and Sal I sites.

Example 2

This example describes cDNA sequencing and library subtraction.Sequencing Template Preparation: Individual colonies were picked and DNAwas prepared either by PCR with M13 forward primers and M13 reverseprimers, or by plasmid isolation. All the cDNA clones were sequencedusing M13 reverse primers.

Q-bot Subtraction Procedure: cDNA libraries subjected to the subtractionprocedure were plated out on 22×22 cm² agar plate at density of about3,000 colonies per plate. The plates were incubated in a 37° C.incubator for 12-24 hours. Colonies were picked into 384-well plates bya robot colony picker, Q-bot (GENETIX Limited). These plates wereincubated overnight at 37° C.

Once sufficient colonies were picked, they were pinned onto 22×22 cm²nylon membranes using Q-bot. Each membrane contained 9,216 colonies or36,864 colonies. These membranes were placed onto agar plate withappropriate antibiotic. The plates were incubated at 37° C. forovernight.

After colonies were recovered on the second day, these filters wereplaced on filter paper pre-wetted with denaturing solution for fourminutes, then were incubated on top of a boiling water bath foradditional four minutes. The filters were then placed on filter paperpre-wetted with neutralizing solution for four minutes. After excesssolution was removed by placing the filters on dry filter papers for oneminute, the colony side of the filters were place into Proteinase Ksolution, incubated at 37° C. for 40-50 minutes. The filters were placedon dry filter papers to dry overnight. DNA was then cross-linked tonylon membrane by UV light treatment. Colony hybridization was conductedas described by Sambrook, et al., (in Molecular

Cloning: A laboratory Manual, 2nd Edition). The following probes wereused in colony hybridization:

1. First strand cDNA from the same tissue as the library was made fromto remove the most redundant clones.

2. 48-192 most redundant cDNA clones from the same library based onprevious sequencing data.

3. 192 most redundant cDNA clones in the entire maize sequence database.

4. ASal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAAAAA AAA, removes clones containing a poly A tail but no cDNA.

5. cDNA clones derived from rRNA.

The image of the autoradiography was scanned into computer and thesignal intensity and cold colony addresses of each colony was analyzed.Re-arraying of cold colonies from 384 well plates to 96 well plates wasconducted using Q-bot.

Example 3

This example describes identification of the gene from a computerhomology search. Gene identities were determined by conducting BLAST(Basic Local Alignment Search Tool; Altschul, et al., (1993) J. Mol.Biol. 215:403-410; see also, National Center for BiotechnologyInformation, National Library of Medicine, Building 38A, Bethesda, Md.,USA) searches under default parameters for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL and DDBJ databases). The cDNAsequences were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithm.

The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States, (1993) J.Nature Genetics 3:266-272) provided by the NCBI. In some cases, thesequencing data from two or more clones containing overlapping segmentsof DNA were used to construct contiguous DNA sequences.

Example 4 Vector Construction and Over Expression of ZM-ERF3 in MaizePHP21751-UBI:ZM-ERF3

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified andligated into a vector containing the maize UBI promoter and PINIIterminator. The gene cassette was then ligated to generate UBIPRO:ZM-ERF3:PINII TERM+35S:BAR:PINII. 35S:BAR is used as a herbicideresistance marker. The expression vector was quality checked byrestriction digestion mapping and transferred into Agrobacteriumtumefaciens LB4404JT by electroporation. This Agrobacterium strain wasused to transform GS3 maize inbred. Molecular analyses on T0 events wereperformed and single copy transgene expressing events were advanced forfurther experiments.

PHP25534-RAB17:ZM-ERF3

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified andligated into a vector containing the maize RAB17 promoter and GZ-W64Aterminator, as well as Gateway (Invitrogen) ATT sites. The entry vectorin combination with a destination vector was used in a single siteGateway (Invitrogen) reaction to generateRAB17:ZM-EFR3:GZ-W64A+UBI:MOPAT:PINII+LTP2:DS-RED:PINII. UBI:MOPAT andLTP2:RFP are used as herbicide resistance and visible markers,respectively. The expression vector was quality checked by restrictiondigestion mapping and transferred into Agrobacterium tumefaciensLB4404JT by electroporation. This Agrobacterium strain was used totransform GS3 maize inbred. Molecular analyses on T0 events wereperformed and single copy transgene expressing events were advanced forfurther experiments. Advancement comprises self-pollination orpollination with the parent genotype and selection for the transgenicprogeny. For example, T1 progeny comprises two doses of the parentalgenotype and may be referred to as D2. Advanced lines may be crossedwith a tester genotype for field evaluation.

Hybrid material representing ten events of PHP25534 was planted inreplicated field trials subjected to drought stress during thegrain-fill stage (i.e., within the R2 to R5 stages as described in How aCorn Plant Develops, Iowa State University of Science and TechnologyCooperative Extension Service Special Report No. 48, Reprinted June1993.) Four of ten events showed statistically significant improvedyield under drought stress as compared to controls. One of those fourevents also demonstrated statistically significant improved performancein seedling assays for drought tolerance and for early vigor underlow-temperature stress.

PHP25536-RYE CBF31 PRO:ZM-ERF3

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified andligated along with the RYE CBF31 promoter (U.S. patent application Ser.No. 12/256,568 filed 23 Oct. 2008) into a vector containing the maizeGZ-W64A terminator, as well as Gateway (Invitrogen) ATT sites. The entryvector in combination with a destination vector was used in a singlesite Gateway (Invitrogen) reaction to generateRAB17:ZM-EFR3:GZ-W64A+UBI:MOPAT:PINII+LTP2:DS-RED:PINII. UBI:MOPAT andLTP2:RFP are used as herbicide resistance and visible markers,respectively. The expression vector was quality checked by restrictiondigestion mapping and transferred into Agrobacterium tumefaciensLB4404JT by electroporation. This Agrobacterium strain was used totransform GS3 maize inbred. Molecular analyses on T0 events wereperformed and single copy transgene expressing events were advanced forfurther experiments. Advancement comprises self-pollination orpollination with the parent genotype and selection for the transgenicprogeny. For example, T1 progeny comprises two doses of the parentalgenotype and may be referred to as D2. Advanced lines may be crossedwith a tester genotype for field evaluation.

Hybrid material representing 9 events of PHP25536 was planted inreplicated field trials subjected to drought stress during thegrain-fill stage. Three of nine events showed statistically significantimproved yield under drought stress as compared to controls. Two ofthose three events, and two additional events, demonstratedstatistically significant improved performance in seedling assays forearly vigor under low-temperature stress.

PHP25537-RYE CBF31 PRO:ZM-ERF3+RYE CBF31 PRO:ZM-CBF2

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified & ligatedalong with the RYE CBF31 promoter into a vector containing the maizeGZ-W64A terminator, as well as Gateway (Invitrogen) ATT sites. The entryvector in combination with a RYE CBF31 PRO:ZM-CBF2 entry vector wereused in a multisite Gateway (Invitrogen) reaction to generate RYE CBF31PRO:ZM-EFR3:GZ-W64A+RYE CBF31:ZM-CBF2:PINII+UBI:MOPAT:PINII. UBI:MOPATis used as a herbicide resistance marker. The expression vector wasquality checked by restriction digestion mapping and transferred intoAgrobacterium tumefaciens LB4404JT by electroporation. ThisAgrobacterium strain was used to transform GS3 maize inbred. Molecularanalyses on T0 events were performed and single copy transgeneexpressing events were advanced for further experiments.

PHP25538-RYE CBF31 PRO:ZM-ERF3+RYE CBF31 PRO:RYE CBF31

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified andligated along with the RYE CBF31 promoter into a vector containing themaize GZ-W64A terminator, as well as Gateway (Invitrogen) ATT sites. Theentry vector in combination with a RYE CBF31 PRO:RYE CBF31 entry vectorwere used in a multisite Gateway (Invitrogen) reaction to generate RYECBF31 PRO:ZM-EFR3:GZ-W64A+RYE CBF31:RYE:CBF31:GZ-W64A+UBI:MOPAT:PINII.UBI:MOPAT is used as a herbicide resistance marker. The expressionvector was quality checked by restriction digestion mapping andtransferred into Agrobacterium tumefaciens LB4404JT by electroporation.This Agrobacterium strain was used to transform GS3 maize inbred.Molecular analyses on T0 events were performed and single copy transgeneexpressing events were advanced for further experiments.

PHP26620-RD29A PRO:ZM-ERF3+RD29A:RYE CBF31

The coding sequence of ZM-ERF3 was amplified by PCR and cloned intopCR2.1 TOPO vector (Invitrogen). ZM-ERF3 was sequence verified andligated along with the RD29A promoter into a vector containing the PINIIterminator, as well as Gateway (Invitrogen) ATT sites. The entry vectorin combination with a RD29A PRO:RYE CBF31 entry vector were used in amultisite Gateway (Invitrogen) reaction to generate RD29APRO:ZM-EFR3:PINII+RD29A:RYE:CBF31:GZ-W64A+UBI:MOPAT:PINII. UBI:MOPAT isused as a herbicide resistance marker. The expression vector was qualitychecked by restriction digestion mapping and transferred intoAgrobacterium tumefaciens LB4404JT by electroporation. ThisAgrobacterium strain was used to transform EF09B maize inbred. Molecularanalyses on T0 events were performed and single copy transgeneexpressing events were advanced for further experiments. Statisticallysignificant yield improvement was observed in one of two events testedunder separate drought stresses at anthesis and during grain-fill.

Example 5 Vector Construction and Gene Silencing of ZM-EIN3 in MaizeUBI:EIN3:PINII RNAi

Two ˜500 base pair (sense and anti-sense) truncated fragments of theZM-EIN3 gene were amplified by PCR and cloned into an Invitrogen TOPOvector. The ZM-EIN3 sense and anti-sense truncated fragments weresequence verified and ligated, along with an ADH1 intron loop sequenceinto a vector containing the maize UBI promoter and PINII terminator, aswell as Gateway (Invitrogen) ATT sites. The entry vector in combinationwith a destination vector was used in a single site Gateway (Invitrogen)reaction to generate UBI:ZM-EIN3:PINIIRNAi+UBI:MOPAT:PINII+LTP2:DS-RED:PINII. UBI:MOPAT and LTP2:RFP are usedas herbicide resistance and visible markers, respectively. Theexpression vector was quality checked by restriction digestion mappingand transferred into Agrobacterium tumefaciens LB4404JT byelectroporation. This Agrobacterium strain was used to transform EFO9Bmaize inbred. Molecular analyses on T0 events were performed and singlecopy transgene expressing events were advanced for further experiments.

Example 6 Sequence Isolation and Endogenous Expression SequenceIsolation

The ethylene signaling genes EIN3 and EIN5 are being used indown-regulation constructs using the RNAi strategy. Two RNAi constructsfor EIN3 and one for EIN5 have been prepared. In the case of EIN3,full-insert sequence from cfp7n.pk010.h4 (PCO642867) has been used togenerate two RNAi constructs with truncated fragments of approximately500 bp at the 5′ end of the coding sequence. One of the two constructsincluded the starting ATG in the RNAi fragment, while the second avoidedthe use of the starting ATG and started immediately after. The EIN5 RNAiconstruct was prepared using approximately 500 bp at the 5′ end of thecoding sequence, starting immediately after the first ATG. Fragments forthe EIN5 RNAi construct were amplified from cfp5n.pk005.c17.f:fis(PCO637491).

Expression Information:

The maize ethylene genes ERF3, EIN3, EIN5, EBF1 and EBF2 are expressedin all tissues in the plant (Table 2). Endogenous expression of ZmERF3is found to be highest in vascular bundles showing an MPSS expressionlevel (Solexa, Hayward, Calif.; Brenner, et al., (2000) NatureBiotechnology 18:630-634) of 628 ppm, while the gene is expressed inpractically all maize tissues (FIG. 1). The highest expression levelsobserved for ZmEIN3, ZmEIN5, ZmEBF1 and ZmEBF2 are, respectively, 1603ppm (internodes), 168 ppm (ear meristem), 560 ppm (root) and 902 ppm(root).

The genes under consideration here also show differential expression inthe presence of stresses or hormones, as indicated in Table 2. ZmERF3 isobserved to be induced by drought in most tissues, although oneexperiment indicated downregulation of the gene in leaves and rootsunder drought. It is also observed to be downregulated by cold stress.The expression of this gene appears to be closely related to the time ofexposure to the stress, as indicated by a microarray experimentconducted to determine the cold-induced time-course of gene expressionin maize seedling leaves. The expression of the gene was found highestat the very early time point of 0.5 hour after exposure to cold stress,and thereafter it declined to normal uninduced levels by 24 hours afterexposure to stress (FIG. 2).

ZmEIN3 is induced by drought stress, and to a lesser extent by coldstress, in aerial tissues, while it appears to be down-regulated bydrought in the root. It also shows a higher expression in response toABA treatment during the early hours (24 h) of ABA exposure. In contrastto this, ZmEIN5 expression is downregulated in most aerial tissues bydrought and upregulated in root. It shows enhanced expression upontreatment with both ABA and ethylene. Expression of ZmEBF1 and ZmEBF2appears to be more or less similarly regulated in the plant. Both areupregulated by drought in aerial tissues and downregulated in roots. Inaddition, ZmEBF1 is induced by cold stress, while ZmEBF2 is induced byboth ABA and ethylene treatment.

Downstream Gene Expression in UBI::Zm ERF3 Transgenic Maize

Constitutive over-expression of ZmERF3 in maize resulted in apleiotropic effect, where the stems of the plants curved as they grew.The plants also exhibited “buggy-whipping”, a phenomenon where the newlyemerging leaves were tightly curled and bent, during the vegetativestage prior to tasseling. However, they recovered from this phenotype asthey grew towards the reproductive stage. Considering that the highestendogenous expression of the gene is in vascular bundles, it is likelythat constitutive overexpression resulted in adversely affectingvascular formation in the stem and this caused the curving of the stalkduring growth. We analyzed two events, namely E3 and E18, of transgenicmaize constitutively expressing ZmERF3 from the UBI promoter, toidentify changes in downstream gene expression. The event, E18, showed apronounced pleiotropic effect, while the event, E3, did not show such aneffect. Transgene expression in event E18 was confirmed to be very highby northern blotting, relative to endogenous levels. As ZmERF3 is atranscription factor, constitutive over-expression of this gene wouldresult in either upregulation or downregulation of genes whoseexpression is regulated at the transcription level by ZmERF3. There wassignificant overlap between the upregulated and downregulated genes inthe two events, with more number of genes showing change in E18 than inE3 (FIG. 3). A list of the genes with known functionality that are up-or down-regulated commonly in the two events is presented in Table 3.Analysis of downstream gene expression indicates the presence of stressand/or ethylene related genes in both the up-regulated anddown-regulated categories. In attempting to overcome the pleiotropiceffect of UBI::ZmERF3, constructs were designed to express the gene fromstress-regulated promoters. Since several stress-related genes aredown-regulated in UBI::ZmERF3 transgenics, one RNAi construct will alsobe prepared to assess the effect of this transcription factor intransgenic stress tolerance.

TABLE 2 Endogenous expression of four ethylene signaling genes asrepresented in MPSS libraries. EXPRESSION DETAILS GENE GeneralExpression Information ZM ERF3 ZM EIN3 ZM EIN5 ZM EBF1 ZM EBF1/2 Tissuespecificity All Tissues All Tissues All Tissues All Tissues All TissuesHighest MPSS expression Vascular Internode Ear Meristem Root (560 ppm)Root (902 ppm) bundles (1603 ppm) (168 ppm) (628 ppm) MPSS TissueLibraries MPSS Expression (ppm) Corn pedicels, drought stressed 162 37 —173  10 Corn pedicels, watered control 62 8 — — — Corn leaf, droughtstressed 38 763 21 65 239  Corn leaf, watered control 65 339 36 — — Cornroot, drought stressed 219 485 53 15 — Corn root, watered control 410650 39 58 27 Corn v5 leaves, ABA treated, 24 hr 68 814 33 — — Corn v5leaves, ABA treated, 48 hr 25 518 79 — 45 Corn v5 leaves, Ethephontreated, 24 hr 26 297 22 — — Corn v5 leaves, Ethephon treated, 48 hr 95521 55 — 59 Corn v5 leaves, control (no hormone treatment) 57 509 20 — 7 Corn seedling, cold stress 28 459 — 30 — Corn seedling, cold-stressrecovery 25 380 — — — Corn seedling, no cold-stress control 109 328 — —— Corn immature ear tips, drought stressed 12 — 68 50 14 Corn immatureear tips, watered control — — 78 — — Corn ear leaf, drought stressed 100—  3 — 44 Corn ear leaf, watered control 5 — 120  27 — Corn 7-DAP apicalkernels, drought stressed 75 — 96 105  24 Corn 7-DAP apical kernels,watered control 29 — 56 — 13 Corn 7-DAP Basal Kernels, drought stressed82 — 128  125  — Corn 7-DAP Basal Kernels, watered control 58 — 99 25 30

TABLE 3-a Top-BLAST hit of genes with known functionality that iscommonly upregulated in both events 3 and 18 of maize transgenicsharboring UBI::ZmERF3. Fold change Accession Gene Name in E18 Q6Z2W4AvrRpt2-induced protein 2-like [Oryza sativa (japonica cultivar-group)]81.584 Q7XLD7 OSJNBa0070C17.11 protein [Oryza sativa (japonicacultivar-group)] 21.523 Q8S0K1 Selenoprotein-like [Oryza sativa(japonica cultivar-group)] 26.790 Q5VQ37 Leaf senescence protein-like[Oryza sativa (japonica cultivar-group)] 6.737 Q08062 Malatedehydrogenase [Zea mays] 9.206 Q0JQR6 Os01g0143500 protein [Oryza sativa(japonica cultivar-group)] 9.720 P42390 Indole-3-glycerol phosphatelyase, chloroplast precursor [Zea mays] 54.913 Q69XR7 Putative acyl-CoAoxidase ACX3 [Oryza sativa (japonica cultivar-group)] 164.089 Q6K4Y6Prefoldin-related K 9.165 Q75I96 Putative receptor-like kinase [Oryzasativa (japonica cultivar-group)] 13.604 Q9XF58 Plasma membraneintrinsic protein [Zea mays] 5.250 Q43417 Peroxidase precursor [Cenchrusciliaris] 8.826 Q0DX49 Putative DNA-3-methyladenine glycosylase [Oryzasativa (japonica cultivar-group)] 10.388 Q6YW60 Zinc finger (C3HC4-typeRING finger) protein-like [Oryza sativa (japonica cultivar-group)]12.170 Q5NA53 Glycogenin-like protein [Oryza sativa (japonicacultivar-group)] 5.573 O48558 60S ribosomal protein L30 [Zea mays]12.328 Q7XLD7 OSJNBa0070C17.11 protein [Oryza sativa (japonicacultivar-group)] 21.523 A2WNN4 Os01g0287400 protein [Oryza sativa(indica cultivar-group)] 41.096 Q75IK0 Putative o-methyltransferase ZRP4[Oryza sativa (japonica cultivar-group)] 17.086

TABLE 3-b Top-BLAST hit of genes with known functionality that iscommonly down-regulated in both events 3 and 18 of maize transgenicsharboring UBI::ZmERF3. Fold change Accession Gene Name in E18 Q6Z6M4Isocitrate lyase [Oryza sativa (japonica cultivar-group)] −16.010 Q75HZ0Putative late embryogenesis abundant protein [Oryza sativa (japonicacultivar-group)] −7.250 Q9AVM3 Cytochrome P450 [Triticum aestivum]−21.360 Q40680 Os07g0614500 protein [Oryza sativa] −11.691 Q10LJ9 Heavymetal-associated domain containing protein, expressed [Oryza sativa]−5.053 Q9ZWI4 ZmGR2c protein [Zea mays] −7.283 Q6J555 MADS16 protein[Dendrocalamus latiflorus] −9.053 A0S6X4 FT-like protein [Hordeumvulgare subsp. vulgare] −11.773 Q5VMA5 Putative lipase [Oryza sativa(japonica cultivar-group)] −6.354 Q9ZSX1 Polyprotein [Zea mays] −6.338O49010 Herbicide safener binding protein [Zea mays] −10.906 Q6L5H6Os05g0537400 protein [Oryza sativa (japonica cultivar-group)] −6.043Q10SX1 Sterol desaturase family protein, expressed [Oryza sativa(japonica cultivar-group)] −5.691 Q2RBL6 Major Facilitator Superfamilyprotein, expressed [Oryza sativa (japonica cultivar-group)] −9.974Q10S44 Basic helix-loop-helix, putative, expressed [Oryza sativa(japonica cultivar-group)] −9.167 Q8W2K4 Cytochrome b5 reductase isoformII [Zea mays] −12.870 Q2R2W1 Adagio-like protein 3 [Oryza sativa] −6.005Q69Y12 Putative aminopeptidase C [Oryza sativa (japonicacultivar-group)] −31.794 Q53JI5 POT family, putative [Oryza sativa(japonica cultivar-group)] −39.680 Q7EYH1 Putative MDR-like ABCtransporter [Oryza sativa (japonica cultivar-group)] −10.733 Q84ZF7Os07g0293000 protein [Oryza sativa (japonica cultivar-group)] −13.433Q69J29 Pectin methylesterase-like protein [Oryza sativa (japonicacultivar-group)] −8.375 Q8RZV3 Zinc finger (C3HC4-type RING finger)-like[Oryza sativa (japonica cultivar-group)] −8.153 Q9LT02 Putativecation-transporting ATPase [Arabidopsis thaliana] −5.999 Q7XIR1 Carbonylreductase-like protein [Oryza sativa (japonica cultivar-group)] −7.132

Example 7 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the Ethylene signaling associated sequence operablylinked to the drought-inducible promoter RAB17 promoter (Vilardell, etal., (1990) Plant Mol Biol 14:423-432) and the selectable marker genePAT, which confers resistance to the herbicide Bialaphos. Alternatively,the selectable marker gene is provided on a separate plasmid.Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue:

The ears are husked and surface sterilized in 30% Clorox® bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA:

A plasmid vector comprising the Ethylene signaling associated sequenceoperably linked to an ubiquitin promoter is made. This plasmid DNA plusplasmid DNA containing a PAT selectable marker is precipitated onto 1.1μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment:

The sample plates are bombarded at level #4 in a particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Subsequent Treatment:

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for increased drought tolerance. Assaysto measure improved drought tolerance are routine in the art andinclude, for example, increased kernel-earring capacity yields underdrought conditions when compared to control maize plants under identicalenvironmental conditions. Alternatively, the transformed plants can bemonitored for a modulation in meristem development (i.e., a decrease inspikelet formation on the ear). See, for example, Bruce, et al., (2002)Journal of Experimental Botany 53:1-13.

Bombardment and Culture Media:

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite® gelling agent (added after bringing to volumewith D-I H₂O); and 8.5 mg/l silver nitrate (added after sterilizing themedium and cooling to room temperature). Selection medium (560R)comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson'sVitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,and 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustmentto pH 5.8 with KOH); 3.0 g/l Gelrite®gelling agent (added after bringingto volume with D-I H₂O); and 0.85 mg/l silver nitrate and 3.0 mg/lbialaphos (both added after sterilizing the medium and cooling to roomtemperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite® gellingagent (added after bringing to volume with D-I H₂O); and 1.0 mg/lindoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing themedium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution(0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxineHCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polishedD-I H₂O after adjusting pH to 5.6); and 6 g/l Bacto™-agar solidifyingagent (added after bringing to volume with polished D-I H₂O), sterilizedand cooled to 60° C.

Example 8 Agrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with an antisensesequence of the Ethylene signaling associated sequence of the presentinvention, preferably the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT Patent Application Publication WO 1998/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the antisense Ethylene signaling associated sequences to atleast one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are preferablyimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Preferably the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants. Plants are monitored and scored for a modulation in meristemdevelopment. For instance, alterations of size and appearance of theshoot and floral meristems and/or increased yields of leaves, flowers,and/or fruits.

Example 9 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing an antisenseEthylene signaling associated sequence operably linked to an ubiquitinpromoter as follows. To induce somatic embryos, cotyledons, 3-5 mm inlength dissected from surface-sterilized, immature seeds of the soybeancultivar A2872, are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising an antisense Ethylenesignaling associated sequence operably linked to the ubiquitin promotercan be isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the vector carrying themarker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 10 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining an antisense Ethylene signaling associated sequences operablylinked to a ubiquitin promoter as follows (see also, European PatentNumber EP 0 486233, herein incorporated by reference, andMalone-Schoneberg, et al., (1994) Plant Science 103:199-207). Maturesunflower seed (Helianthus annuus L.) are dehulled using a singlewheat-head thresher. Seeds are surface sterilized for 30 minutes in a20% Clorox® bleach solution with the addition of two drops of Tween® 20per 50 ml of solution. The seeds are rinsed twice with sterile distilledwater.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al. (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant., 15:473-497), Shepard's vitamin additions (Shepard, (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/lsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA₃), pH 5.6, and 8 g/l Phytagar(Invitrogen, Carlsbad, Calif.).

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the ethylene signaling associated geneoperably linked to the ubiquitin promoter is introduced intoAgrobacterium strain EHA105 via freeze-thawing as described by Holsters,et al., (1978) Mol. Gen. Genet. 163:181-187. This plasmid furthercomprises a kanamycin selectable marker gene (i.e, nptII). Bacteria forplant transformation experiments are grown overnight (28° C. and 100 RPMcontinuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriateantibiotics required for bacterial strain and binary plasmidmaintenance. The suspension is used when it reaches an OD₆₀₀ of about0.4 to 0.8. The Agrobacterium cells are pelleted and resuspended at afinal OD₆₀₀ of 0.5 in an inoculation medium comprised of 12.5 mM MES pH5.7, 1 gm/l NH₄Cl, and 0.3 gm/l MgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for a modulation in meristemdevelopment (i.e., an alteration of size and appearance of shoot andfloral meristems).

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%Gelrite® gelling agent, pH 5.6) and grown under conditions described forexplant culture. The upper portion of the seedling is removed, a 1 cmvertical slice is made in the hypocotyl, and the transformed shootinserted into the cut. The entire area is wrapped with Parafilm®flexible film to secure the shoot. Grafted plants can be transferred tosoil following one week of in vitro culture. Grafts in soil aremaintained under high humidity conditions followed by a slowacclimatization to the greenhouse environment. Transformed sectors of T₀plants (parental generation) maturing in the greenhouse are identifiedby NPTII ELISA and/or by Ethylene signaling associated activity analysisof leaf extracts while transgenic seeds harvested from NPTII-positiveT_(o) plants are identified by Ethylene signaling associated activityanalysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Clorox®bleach solution with the addition of two to three drops of Tween® 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagar(Invitrogen, Carlsbad, Calif.) at pH 5.6) for 24 hours under the dark.The primary leaves are removed to expose the apical meristem, around 40explants are placed with the apical dome facing upward in a 2 cm circlein the center of 374M (GBA medium with 1.2% Phytagar (Invitrogen,Carlsbad, Calif.)), and then cultured on the medium for 24 hours in thedark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 pg/I kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD₆₀₀.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for a modulation in meristem development (i.e., an alterationof size and appearance of shoot and floral meristems). After positiveexplants are identified, those shoots that fail to exhibit modifiedEthylene signaling associated activity are discarded, and every positiveexplant is subdivided into nodal explants. One nodal explant contains atleast one potential node. The nodal segments are cultured on GBA mediumfor three to four days to promote the formation of auxiliary buds fromeach node. Then they are transferred to 374C medium and allowed todevelop for an additional four weeks. Developing buds are separated andcultured for an additional four weeks on 374C medium. Pooled leafsamples from each newly recovered shoot are screened again by theappropriate protein activity assay. At this time, the positive shootsrecovered from a single node will generally have been enriched in thetransgenic sector detected in the initial assay prior to nodal culture.

Recovered shoots positive for modified Ethylene signaling associatedexpression are grafted to Pioneer Hybrid 6440 in vitro-grown sunflowerseedling rootstock. The rootstocks are prepared in the following manner.Seeds are dehulled and surface-sterilized for 20 minutes in a 20%Clorox® bleach solution with the addition of two to three drops ofTween® 20 per 100 ml of solution, and are rinsed three times withdistilled water. The sterilized seeds are germinated on the filtermoistened with water for three days, then they are transferred into 48medium (half-strength MS salt, 0.5% sucrose, 0.3% Gelrite® gelling agentpH 5.0) and grown at 26° C. under the dark for three days, thenincubated at 16-hour-day culture conditions. The upper portion ofselected seedling is removed, a vertical slice is made in eachhypocotyl, and a transformed shoot is inserted into a V-cut. The cutarea is wrapped with Parafilm® flexible film. After one week of cultureon the medium, grafted plants are transferred to soil. In the first twoweeks, they are maintained under high humidity conditions to acclimatizeto a greenhouse environment.

Example 11 Rice Tissue Transformation

One method for transforming DNA into cells of higher plants that isavailable to those skilled in the art is high-velocity ballisticbombardment using metal particles coated with the nucleic acidconstructs of interest (see, Klein, et al., Nature (1987) (London)327:70-73 and see, U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He(BioRAD Laboratories, Hercules, Calif.) is used for thesecomplementation experiments. The particle bombardment technique is usedto transform the Ethylene signaling associated mutants and wild typerice with DNA fragments

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic isused as the selectable marker for rice transformation. In the vector,pML18, the Hpt II gene was engineered with the 35S promoter fromCauliflower Mosaic Virus and the termination and polyadenylation signalsfrom the octopine synthase gene of Agrobacterium tumefaciens. pML18 wasdescribed in WO 1997/47731, which was published on Dec. 18, 1997, thedisclosure of which is hereby incorporated by reference.

Embryogenic callus cultures derived from the scutellum of germinatingrice seeds serve as source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is the transferred to CMmedia (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al.,1985, Sci. Sinica 18: 659-668). Callus cultures are maintained on CM byroutine sub-culture at two week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman® #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C. for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Each genomic DNA fragment is co-precipitated with pML18 containing theselectable marker for rice transformation onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs are added to 50 μl aliquot of goldparticles that have been resuspended at a concentration of 60 mg ml⁻¹.Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a0.1 M solution) are then added to the gold-DNA suspension as the tube isvortexing for 3 min. The gold particles are centrifuged in a microfugefor 1 sec and the supernatant removed. The gold particles are thenwashed twice with 1 ml of absolute ethanol and then resuspended in 50 μlof absolute ethanol and sonicated (bath sonicator) for one second todisperse the gold particles. The gold suspension is incubated at −70° C.for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six μl of the DNA-coated gold particles are then loadedonto mylar macrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Two to four plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates are incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% Gelrite® gelling agent +50 ppmhyg B) for 2 weeks in the dark at 25° C. After 2 weeks the callus istransferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3%sucrose, 0.4% Gelrite® gelling agent +50 ppm hyg B) and placed undercool white light (˜40 μEm⁻² s⁻¹) with a 12 hr photo period at 25° C. and30-40% humidity. After 2-4 weeks in the light, callus begin to organize,and form shoots. Shoots are removed from surrounding callus/media andgently transferred to RM3 media (½×MS salts, Nitsch and Nitsch vitamins,1% sucrose +50 ppm hygromycin B) in Phytatray™ disposable plant cellculture vessels (Sigma Chemical Co., St. Louis, Mo.) and incubation iscontinued using the same conditions as described in the previous step.

Plants are transferred from RM3 to 4″ pots containing Scotts MetroMix®350 growing medium after 2-3 weeks, when sufficient root and shootgrowth have occurred. The seed obtained from the transgenic plants isexamined for genetic complementation of the Ethylene signalingassociated mutation with the wild-type genomic DNA containing theEthylene signaling associated gene.

Example 12 Variants of Ethylene Signaling Associated Sequences

A. Variant Nucleotide Sequences of Ethylene Signaling AssociatedProteins that do Not Alter the Encoded Amino Acid Sequence

The Ethylene signaling associated nucleotide sequences are used togenerate variant nucleotide sequences having the nucleotide sequence ofthe open reading frame with about 70%, 75%, 80%, 85%, 90% and 95%nucleotide sequence identity when compared to the starting unaltered ORFnucleotide sequence of the corresponding SEQ ID NO. These functionalvariants are generated using a standard codon table. While thenucleotide sequence of the variants are altered, the amino acid sequenceencoded by the open reading frames do not change.

B. Variant Amino Acid Sequences of Ethylene Signaling AssociatedPolypeptides

Variant amino acid sequences of the Ethylene signaling associatedpolypeptides are generated. In this example, one amino acid is altered.Specifically, the open reading frames are reviewed to determine theappropriate amino acid alteration. The selection of the amino acid tochange is made by consulting the protein alignment (with the otherorthologs and other gene family members from various species). An aminoacid is selected that is deemed not to be under high selection pressure(not highly conserved) and which is rather easily substituted by anamino acid with similar chemical characteristics (i.e., similarfunctional side-chain). Using the protein alignment, an appropriateamino acid can be changed. Once the targeted amino acid is identified,the procedure outlined in the following section C is followed. Variantshaving about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequenceidentity are generated using this method.

C. Additional Variant Amino Acid Sequences of Ethylene SignalingAssociated Polypeptides

In this example, artificial protein sequences are created having 80%,85%, 90%, and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom the alignment and then the judicious application of an amino acidsubstitutions table. These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among Ethylene signaling associatedprotein or among the other Ethylene signaling associated polypeptides.Based on the sequence alignment, the various regions of the Ethylenesignaling associated polypeptide that can likely be altered arerepresented in lower case letters, while the conserved regions arerepresented by capital letters. It is recognized that conservativesubstitutions can be made in the conserved regions below withoutaltering function. In addition, one of skill will understand thatfunctional variants of the Ethylene signaling associated sequence of theinvention can have minor non-conserved amino acid alterations in theconserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 4.

TABLE 4 Substitution Table Rank of Amino Strongly Similar and Order toAcid Optimal Substitution Change Comment I L, V 1 50:50 substitution LI, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C, and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal. Then leucine,and so on down the list until the desired target it reached. Interimnumber substitutions can be made so as not to cause reversal of changes.The list is ordered 1-17, so start with as many isoleucine changes asneeded before leucine, and so on down to methionine. Clearly many aminoacids will in this manner not need to be changed. L, I and V willinvolve a 50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof the Ethylene signaling associated polypeptides are generating havingabout 80%, 85%, 90% and 95% amino acid identity to the startingunaltered ORF nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7 or 9.

Example 13 Transgenic Maize Plants

T₀ transgenic maize plants containing the Ethylene signaling associatedconstruct under the control of a promoter were generated. These plantswere grown in greenhouse conditions, under the FASTCORN system, asdetailed in US Patent Application Publication Number 2003/0221212, U.S.patent application Ser. No. 10/367,417.

Each of the plants was analyzed for measurable alteration in one or moreof the following characteristics in the following manner:

T₁ progeny derived from self fertilization of each T_(o) plantcontaining a single copy of each Ethylene signaling associated constructthat were found to segregate 1:1 for the transgenic event were analyzedfor improved growth rate in low KNO₃. Growth was monitored up toanthesis when cumulative plant growth, growth rate and ear weight weredetermined for transgene positive, transgene null, and non-transformedcontrol events. The distribution of the phenotype of individual plantswas compared to the distribution of a control set and to thedistribution of all the remaining treatments. Variances for each setwere calculated and compared using an F test, comparing the eventvariance to a non-transgenic control set variance and to the pooledvariance of the remaining events in the experiment. The greater theresponse to KNO₃, the greater the variance within an event set and thegreater the F value. Positive results will be compared to thedistribution of the transgene within the event to make sure the responsesegregates with the transgene.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A recombinant expression cassette comprising apolynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide which has at least 95% sequenceidentity, as determined by the GAP algorithm under default parameters,to a polypeptide of SEQ ID NO: 8, 4 or 2; (b) a polynucleotide encodingthe polypeptide of SEQ ID NO: 8, 4 or 2; (c) a polynucleotide of SEQ IDNO: 7, 3 or 1; (d) a polynucleotide which has at least 95% sequenceidentity, as determined by the GAP algorithm under default parameters,to the full length of SEQ ID NO: 7, 3 or 1; (e) a polynucleotide whichhas at least 95% sequence identity, as determined by the GAP algorithmunder default parameters, to the full length of the coding sequence ofSEQ ID NO: 7, 3 or 1; and (f) a polynucleotide which is complementary tothe polynucleotide of (a), (b), (c), (d), or (e), wherein saidpolynucleotide is operably linked, in sense or anti-sense orientation,to a promoter.
 2. A host cell comprising the recombinant expressioncassette of claim
 1. 3. A transgenic plant comprising the recombinantexpression cassette of claim
 1. 4. The transgenic plant of claim 3,wherein said plant is a monocot.
 5. The transgenic plant of claim 3,wherein said plant is selected from the group consisting of: maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley and millet.
 6. A seed from the transgenic plant of claim 3,wherein said seed comprises the recombinant expression cassette ofclaim
 1. 7. A method of modulating ethylene response in a plant,comprising: (a) introducing into a plant cell a recombinant expressioncassette comprising a promoter operably linked to a polynucleotide whichhas at least 95% sequence identity, as determined by the GAP algorithmunder default parameters, to the coding sequence of SEQ ID NO: 3; (b)culturing the plant cell under plant cell growing conditions; (c)regenerating a plant from said cultured plant cell; and (d) inducingexpression of said polynucleotide by drought or cold for a timesufficient to modulate the level of EBF1 in said plant.
 8. The method ofclaim 7, wherein the level or activity of EBF1 in aerial portions of theplant is increased relative to a control.
 9. The method of claim 7,wherein the plant is selected from the group consisting of: maize,soybean, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet,peanut and cocoa.
 10. The method of claim 7, wherein said plant cell isfrom a monocot.
 11. A transgenic plant produced by the method of claim7, wherein the plant has decreased ethylene sensitivity when compared toa control plant.
 12. A method of modulating ethylene response in aplant, comprising: (a) introducing into a plant cell a recombinantexpression cassette comprising a promoter operably linked to apolynucleotide complementary to SEQ ID NO: 7; (b) culturing the plantcell under plant cell growing conditions; (c) regenerating a plant fromsaid cultured plant cell; and (c) inducing expression of saidpolynucleotide by drought, ABA, or ethylene for a time sufficient tomodulate the level of EIN5 in said plant.
 13. The method of claim 12,wherein the level or activity of EIN5 in aerial parts of the plant isdecreased, relative to a control.
 14. The method of claim 12, whereinthe plant is selected from the group consisting of: maize, soybean,sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, peanutand cocoa.
 15. The method of claim 12, wherein said plant cell is from amonocot.
 16. A transgenic plant produced by the method of claim 12,wherein the plant has decreased ethylene sensitivity when compared to acontrol plant.